Microreactor particles for modeling of infectious bone defects, methods of making and use

Abstract

The application discloses a kind of microreactor particles for infectious bone defect modeling and preparation method and application, it is related to medical animal model technical field.The microreactor particle is achieved space fixation by preventing the escape limited domain hydrogel shell, and realizes local real pathogen planting by pathogen nutrition-adhesion microdomain layer and limited pathogen planting layer, and realizes biological membrane sample barrier formation by biomimetic biological membrane ion condensation layer, and realizes chronic inflammation sample metabolic environment maintenance by immune metabolism regulation layer, and low oxygen acidification microenvironment regulation layer simulates infection focus metabolic imbalance, and realizes infection and bone absorption synchronization by osteoclast activation regulation layer, and acid-sensitive bone mineral sacrifice nucleus simulates bone mineral destruction, finally in bone defect local formation has the infection characteristics and bone destruction characteristics of infectious bone defect lesion, and limit pathogenic microorganism and inflammatory stimulation to non-target area diffusion, improve the stability, consistency and repeatability of infectious bone defect animal model.

Description

Technical Field

[0001] This invention relates to the field of medical animal model technology, specifically to a microreactor particle for modeling infectious bone defects, its preparation method, and its application. Background Technology

[0002] Infected bone defects are a challenging type of disease in orthopedic clinics, commonly seen in open fractures, infections following internal fixation or joint replacement surgery, post-traumatic osteomyelitis, and secondary infections following debridement of bone tumors or osteonecrosis. These lesions typically involve simultaneous pathological processes such as pathogen colonization, local chronic inflammation, bone necrosis, increased bone resorption, and inhibited bone regeneration. Treatment is lengthy, recurrence rates are high, and they can easily lead to serious consequences such as nonunion, implant failure, and limb dysfunction.

[0003] Infectious bone defects are not simply bone tissue losses; their core pathological feature lies in the coupling between the infection microenvironment and the bone destruction process. On the one hand, pathogenic microorganisms can adhere to and colonize the bone defect area or implant surface, forming biofilm-like structures that enhance their resistance to host immune clearance and drug intervention. On the other hand, the infection focus is often accompanied by changes such as hypoxia, acidification, increased reactive oxygen species, continuous release of inflammatory factors, and immune metabolic disorders. These factors can further promote osteoclast activation, bone mineral matrix erosion, and bone tissue destruction. Therefore, establishing stable, reproducible animal models that reflect the true pathological characteristics of infectious bone defects is of great significance for studying the mechanisms of infectious bone defects, evaluating anti-infective materials, screening bone repair materials, and developing related treatment strategies.

[0004] Existing animal models of infectious bone defects typically employ methods such as directly inoculating the bone defect area with pathogenic microorganisms, implanting contaminated metal needles or internal fixation devices, or using bacterial-loaded bone cement, bacterial-loaded sponges, or bacterial-loaded hydrogels. While these methods can induce local infection to some extent, they still have several limitations.

[0005] First, direct inoculation of free pathogens is easily affected by local bleeding, tissue fluid flushing, individual animal immune differences, and surgical procedures, resulting in unstable retention and colonization of pathogens at bone defect sites, thus causing large fluctuations in model success rate and infection severity.

[0006] Secondly, contaminated implants or ordinary bacterial carriers mainly serve as carriers of pathogenic microorganisms, lacking a systematic simulation of key pathological processes in real infectious bone defects, such as biofilm barriers, hypoxia and acidification, immune metabolic imbalance, and infection-bone resorption coupling. This can easily lead to problems such as the presence of local infection but insufficient bone destruction, or the presence of mechanical bone defects but unstable infection pathology.

[0007] Furthermore, traditional bacterial carrier materials are mostly single-carrier structures, which allow pathogenic microorganisms to easily spread in a free state to surrounding soft tissues or areas far from bone defects, making it difficult to control the scope of infection and affecting the locality, safety, and reproducibility of animal models.

[0008] In addition, existing models often fail to coordinate processes such as pathogen colonization, biofilm formation, maintenance of chronic inflammation, formation of hypoxic acidification microenvironment, osteoclast activation, and bone mineral destruction within the same local space, thus failing to fully simulate the continuous pathological chain of the development of real infectious bone defects. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a microreactor particle for modeling infectious bone defects, its preparation method, and its application. This microreactor particle for modeling infectious bone defects can achieve localized colonization of pathogenic microorganisms within the pre-fabricated bone defect area, while simultaneously promoting the formation of biofilm-like structures, maintaining the infectious inflammatory microenvironment, inducing a hypoxic acidification state, promoting the coupling of infection and osteoclast processes, and limiting the diffusion of pathogenic microorganisms and inflammatory stimuli to non-target areas, thereby improving the stability, consistency, and reproducibility of the infectious bone defect animal model.

[0010] According to one aspect of the present invention, a method for preparing microreactor particles for modeling infectious bone defects is provided, comprising: S1, preparation of acid-sensitive bone mineral sacrificial cores; Calcium phosphate-based mineral particles containing calcium, phosphorus, magnesium, strontium, and carbonate components were prepared as acid-sensitive bone mineral sacrificial cores. S2, constructing a pathogen nutrition-adhesion microdomain layer; Methacrylamide gelatin, hyaluronic acid-dopamine, fibrinogen mimic peptide, collagen-binding peptide, glucose and calcium chloride were dissolved in phosphate buffer to obtain pathogen nutrient-adhesion microdomain precursor solution; acid-sensitive bone mineral sacrificial cores were added to the pathogen nutrient-adhesion microdomain precursor solution, and then a photoinitiator was added. After photocrosslinking, the particles were collected to obtain intermediate particle A. S3, constructing a restricted pathogen colonization layer; Sodium alginate, carboxymethyl chitosan, oxidized dextran and gelatin were dissolved in phosphate buffer to obtain a colonization layer substrate solution; Staphylococcus aureus was evenly dispersed in the colonization layer substrate solution, intermediate particles A were added, and calcium chloride solution was added dropwise to form a restricted pathogen colonization layer containing pathogenic microorganisms. The particles were washed and collected to obtain intermediate particles B. S4, constructing a biomimetic biological membrane ion condensation layer; Sodium alginate, polyglutamic acid, chondroitin sulfate, and sodium deoxyribonucleic acid were dissolved in HEPES buffer to obtain a polyanionic solution; poly-L-lysine hydrobromide, calcium chloride, and magnesium chloride were dissolved in HEPES buffer to obtain a cationic bridging solution; intermediate particle B was added to the polyanionic solution, followed by the cationic bridging solution, to form a biomimetic biomembrane ion condensation layer; the particles were washed and collected to obtain intermediate particle C; S5, constructing an immune metabolic regulation layer; Polylactic acid-glycolic acid copolymer was dissolved in dichloromethane to obtain an organic phase; sodium lactate and sodium succinate were dissolved in water to obtain an aqueous phase; the aqueous phase was added to the organic phase and emulsified to form a primary emulsion; the primary emulsion was added to a polyvinyl alcohol aqueous solution for further emulsification, allowing the dichloromethane to evaporate; the microparticles were collected, washed, and dried to obtain lactate-succinate sustained-release microparticles; carboxymethyl chitosan and oxidized dextran were dissolved separately in phosphate buffer, mixed, and then added to the lactate-succinate sustained-release microparticles; the pH was adjusted to obtain an immunometabolic reprogramming coating solution; intermediate particle C was added to the immunometabolic reprogramming coating solution to form an immunometabolic regulation layer; the particles were washed and collected to obtain intermediate particle D; S6, constructing a osteoclast activation regulation layer; Hydrogenated soybean phospholipids, cholesterol, and DSPE-PEG-COOH were dissolved in a mixed solvent of chloroform and methanol. The mixture was evaporated under reduced pressure to form a lipid film. After drying, the film was hydrated with phosphate buffer and homogenized to obtain an exosome-inspired vesicle suspension. EDC and NHS were added to the obtained exosome-inspired vesicle suspension to adjust the pH to acidic and activate the carboxyl groups. Bone-targeting peptides and osteoclast-inducing mimic peptides were added to adjust the pH to weakly alkaline. After the reaction, the film was dialyzed to obtain an osteoclast-inducing exosome-inspired vesicle suspension. Intermediate particle D was added to the osteoclast-inducing exosome-inspired vesicle suspension and incubated to form an osteoclast-activating regulatory layer. The particles were washed and collected to obtain intermediate particle E. S7, constructing a low-oxygen acidification microenvironment regulation layer; Poly-L-histidine hydrochloride was dissolved in MES buffer, the pH was adjusted, and glucose oxidase, MIL-100(Fe) nanoparticles, branched polyethyleneimine and gluconolactone were added to obtain a hypoxia-acidified microenvironment conditioning layer coating solution. Intermediate particle E was added to the hypoxia-acidified microenvironment conditioning layer coating solution to form a hypoxia-acidified microenvironment conditioning layer. The particles were washed and collected to obtain intermediate particle F. S8, constructing an escape-proof confined hydrogel shell; Sulfobetaine methacrylate, β-cyclodextrin-modified hyaluronic acid, adamantane-modified gelatin, and methoxy polyethylene glycol phosphonic acid were dissolved in phosphate buffer to obtain a shell precursor solution. N,N'-methylenebisacrylamide, ammonium persulfate, and intermediate particles F were added to the shell precursor solution, followed by the addition of N,N,N',N'-tetramethylethylenediamine. After the reaction was completed, the mixture was washed to obtain wet pathogen-confined infectious bone defect modeling microreactor particles.

[0011] Optionally, after obtaining the wet pathogen-confined infectious bone defect modeling microreactor particles in S8, the method further includes: S9, dispersing the wet pathogen-confined infectious bone defect modeling microreactor particles in sterile phosphate buffer containing a lyophilization protectant, pre-freezing and then freeze-drying to obtain dry pathogen-confined infectious bone defect modeling microreactor particles.

[0012] Optionally, S1, the preparation of acid-sensitive bone mineral sacrificial cores specifically includes: dissolving calcium nitrate tetrahydrate, magnesium chloride hexahydrate, and strontium nitrate in water to obtain a calcium-magnesium-strontium mixed solution; dissolving diammonium hydrogen phosphate, sodium bicarbonate, and sodium polyaspartate in water to obtain a phosphate-carbonate solution; adding the obtained phosphate-carbonate solution dropwise to the calcium-magnesium-strontium mixed solution, adjusting the pH, allowing the mixture to stand and age after the reaction, collecting the precipitate, washing and drying it to obtain acid-sensitive bone mineral sacrificial core powder.

[0013] Optionally, in S2, the fibrinogen mimic peptide is Cys-Gly-Pro-Arg-Pro-Pro, and the collagen-binding peptide is Thr-Lys-Lys-Thr-Leu-Arg-Thr.

[0014] Optionally, in S3, the *Staphylococcus aureus* strain is in the logarithmic growth phase, washed with sterile phosphate buffer and resuspended, resulting in a bacterial suspension concentration of 1 × 10⁻⁶. 8 CFU / mL, and add it to the planting layer substrate solution at 5%~10% of the substrate solution volume.

[0015] Optionally, in S6, the bone-targeting peptide is an octameric aspartic peptide, and the osteoclast-inducing mimic peptide is a human parathyroid hormone 1-34 fragment, with a mass ratio of 0.5 to 2.

[0016] Optionally, in S8, the adamantane-modified gelatin is prepared by dissolving gelatin in MES buffer to obtain a gelatin solution; taking 1-adamantane carboxylic acid, dissolving it in anhydrous dimethyl sulfoxide, then adding EDC·HCl and NHS, and activating it at room temperature to obtain an activated 1-adamantane carboxylic acid solution; adding the obtained activated 1-adamantane carboxylic acid solution dropwise to the gelatin solution, and after the reaction is complete, dialysis and drying to obtain adamantane-modified gelatin.

[0017] According to another aspect of the present invention, a microreactor particle for modeling infectious bone defects is provided, which is prepared by the method for preparing microreactor particles for modeling infectious bone defects as described above.

[0018] Optionally, the microreactor particles used for modeling infectious bone defects can be further dispersed in an injectable externally sealed hydrogel carrier to form a particle-gel composite system.

[0019] According to another aspect of the invention, the application of the microreactor particles described above for modeling infectious bone defects in constructing infectious bone defect-like lesion materials is provided.

[0020] The beneficial effects of this invention are: (1) This invention, through a restricted pathogen colonization layer, an anti-escape confinement hydrogel shell, and an externally sealed hydrogel carrier, primarily confines bone infection-related pathogens within the pre-fabricated bone defect area, reducing the possibility of pathogens spreading to surrounding soft tissues in a free state. Compared with traditional methods of directly inoculating pathogens or using contaminated implants for modeling, this invention is beneficial in improving the retention and colonization stability of pathogens in the bone defect area, thereby enhancing the locality, controllability, and repeatability of model establishment.

[0021] (2) This invention does not simply carry pathogenic microorganisms, but rather, through a pathogen nutrition-adhesion microdomain layer, a biomimetic biomembrane ion aggregation layer, an immune metabolism regulation layer, and a hypoxia-acidification microenvironment regulation layer, it synergistically forms pathogen adhesion and colonization, a biomembrane-like matrix barrier, continuous inflammatory stimulation, and a local metabolic imbalance state in the bone defect area. This allows the animal model to more closely resemble the pathological characteristics of clinical infectious bone defects, which include pathogen colonization, biomembrane-like structure formation, persistent inflammation, and local microenvironment disorder.

[0022] (3) This invention promotes the correlation between infectious inflammation and local bone resorption by regulating osteoclast activation, and simulates the bone mineral matrix damage process in infectious bone defects by the dissolution behavior of acid-sensitive bone mineral sacrificial nuclei in an acidic inflammatory environment. Under the action of the material of this invention, the prefabricated bone defect area can exhibit infectious bone defect-like changes such as continuous bacterial colonization, inflammatory infiltration, decreased bone density, and bone defect expansion, thereby improving the pathological consistency and experimental reproducibility of the animal model. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 A scanning electron microscope (SEM) image of the sacrificial nucleus of acid-sensitive bone minerals; Figure 2 This is a scanning electron microscope (SEM) image of intermediate particle A; Figure 3 Image of intermediate particle B using a scanning electron microscope (SEM). Figure 4 Image of intermediate particle C using scanning electron microscopy (SEM); Figure 5 This is a scanning electron microscope (SEM) image of intermediate particles. Figure 6 Image of intermediate particles using scanning electron microscopy (SEM); Figure 7 Image of intermediate particles using a scanning electron microscope (SEM); Figure 8 Scanning electron microscope images of microreactor particles used to model pathogen-confined infectious bone defects; Figure 9 In vitro validation data for pathogen confined colonization and biomimetic biofilm formation; Figure 10 Ca2+ as a sacrificial nucleus for acid-sensitive bone minerals under different pH conditions 2+ Cumulative release results; Figure 11 Mg as a sacrificial nucleus for acid-sensitive bone minerals under different pH conditions 2+ Cumulative release results; Figure 12 Sr, a sacrificial nucleus for acid-sensitive bone minerals under different pH conditions 2+ Cumulative release results; Figure 13 Images of rat leg bones after implantation of microreactor particles; Figure 14 Micro-CT images of rat leg bones after implantation of microreactor particles; Figure 15 A diagram showing the local infection in a rat leg bone model 28 days after microreactor particle implantation; Figure 16 A graph showing the effect of microreactor particles in constructing infectious bone defect-like lesions in vivo. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0025] Unless otherwise specified, all conditions in the examples were performed under standard conditions or according to the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are commercially available standard products. Unless otherwise stated, all technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art.

[0026] This invention provides microreactor particles for modeling infectious bone defects, a preparation method, and applications. The microreactor particles are suitable for implantation into pre-formed bone defect cavities. Through local pathogen colonization, pre-organization of biofilms, hypoxia-acidification self-feedback, immune metabolic reprogramming, and infection-bone resorption coupling, the pre-fabricated mechanical bone defect gradually transforms into an infectious bone defect lesion with real infectious pathological characteristics.

[0027] The microreactor particles, from the inside out, consist of: an acid-sensitive bone mineral sacrificial core; a pathogen nutrition-adhesion microdomain layer; a restricted pathogen colonization layer; a biomimetic biomembrane ion aggregation layer; an immune metabolism regulation layer; a osteoclast activation regulation layer; a hypoxia acidification microenvironment regulation layer; and an anti-escape confinement hydrogel shell layer.

[0028] Among them, the acid-sensitive bone mineral sacrificial core is used to induce mineral dissolution in an infectious acidified environment; the pathogen nutrition-adhesion microdomain layer is used to promote the adhesion and initial colonization of bone infection-related pathogens in the local bone defect area; the restricted pathogen colonization layer is used to confine bone infection-related pathogens to the local microcavities or gel network of the microreactor particles; the biomimetic biomembrane ion aggregation layer is used to promote the formation of local biomembrane-like structures; the immune metabolism regulation layer is used to create a chronic inflammatory-like metabolic environment; the osteoclast activation regulation layer is used to promote the synchronous occurrence of infectious inflammation and bone resorption processes; the hypoxia acidification microenvironment regulation layer is used to create an infection focus-like hypoxia and acidification state; and the escape-prevention confining hydrogel shell is used to reduce the risk of pathogens, inflammatory stimuli, and particle bulk spreading outside the bone defect area.

[0029] It should be further clarified that the terms "confined," "prevents escape," "biofilm-like," "infection-like," and "hypoxia-acidification" used in this invention refer to the enhanced effects on the local retention of pathogenic microorganisms, the formation of infection-related barrier structures, and the simulation of infection-foci-like microenvironments compared to traditional free-borne or ordinary bacterial carrier materials. They do not imply absolute blocking of pathogenic microorganism diffusion, local oxidative stress, or acidification reactions, or complete equivalence to natural lesions. The dosage of active components in each functional layer can be routinely optimized based on the target pathogenic microorganism species, animal model type, and expected infection level to maintain a local infection-like microenvironment while avoiding complete eradication of the target pathogenic microorganism.

[0030] Example 1: Pathogen-confined microreactor particles for modeling infectious bone defects, i.e., the preparation of the microreactor particles used for modeling infectious bone defects.

[0031] This embodiment uses 1.00g of acid-sensitive bone mineral sacrificial core as the starting amount to illustrate the preparation process of microreactor particles for pathogen-confined infectious bone defect modeling.

[0032] I. Preparation of acid-sensitive bone mineral sacrificial cores: Step 1.1: Prepare a calcium-magnesium-strontium mixed solution; Take 2.36 g of calcium nitrate tetrahydrate, 0.20 g of magnesium chloride hexahydrate and 0.10 g of strontium nitrate, add them to 100 mL of deionized water, and stir to dissolve at room temperature. The stirring speed is 500 rpm and the stirring time is 30 min to obtain a calcium-magnesium-strontium mixed solution.

[0033] Step 1.2: Prepare a phosphate-carbonate solution; Take 0.79 g of diammonium hydrogen phosphate, 0.42 g of sodium bicarbonate and 0.30 g of sodium polyaspartate, add them to 150 mL of deionized water, and stir to dissolve at room temperature. The stirring speed is 500 rpm and the stirring time is 30 min to obtain a phosphate-carbonate solution.

[0034] Step 1.3: Mineralization and precipitation; The calcium-magnesium-strontium mixed solution obtained in step 1.1 was placed in a reaction vessel at 25°C and stirred at 600 rpm. The phosphate-carbonate solution obtained in step 1.2 was added dropwise to the calcium-magnesium-strontium mixed solution at a rate of 2 mL / min. During the addition, the pH of the system was adjusted with ammonia water and maintained at 8.5–9.0. After the addition was complete, the reaction was continued at 25°C for 2 h with stirring at 600 rpm to obtain a milky white mineralized suspension.

[0035] Step 1.4: Aging, washing, and drying; The obtained mineralized suspension was allowed to stand for 12 hours to age. After aging, it was centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The resulting precipitate was washed three times with 100 mL of deionized water each time, and then washed once with anhydrous ethanol. After each wash, it was centrifuged at 3000 rpm for 10 minutes. The washed precipitate was pre-frozen at -20℃ for 4 hours, and then freeze-dried for 24 hours to obtain acid-sensitive bone mineral sacrificial core powder, such as... Figure 1 As shown.

[0036] The acid-sensitive bone mineral sacrificial core is a calcium phosphate-based mineral particle containing calcium, phosphorus, magnesium, strontium, and carbonate components. This type of mineral particle is more prone to proton-induced mineral dissolution in an acidic inflammatory microenvironment, releasing calcium, magnesium, and strontium ions. It can be used to simulate the process of bone mineral matrix being destroyed by an acidic environment in areas of infectious bone defects.

[0037] II. Construction of the Pathogen Nutrition-Adhesion Microdomain Layer: Step 2.1: Prepare the adhesion microdomain precursor solution; Take 0.40 g of methacrylamide gelatin, 0.20 g of hyaluronic acid-dopamine, 10 mg of fibrinogen mimic peptide, 10 mg of collagen-binding peptide, 0.10 g of glucose, and 0.05 g of calcium chloride, and add them to 100 mL of phosphate buffer. Dissolve by stirring at 37 °C and 400 rpm for 1 h to obtain the pathogen nutrient-adhesion microdomain precursor solution.

[0038] The fibrinogen mimic peptide is a short peptide containing a GPRP or GPRPP motif, which in this embodiment is Cys-Gly-Pro-Arg-Pro-Pro. The collagen-binding peptide is Thr-Lys-Lys-Thr-Leu-Arg-Thr, abbreviated as TKKTLRT (both can be obtained by commercially available custom synthesis or prepared using conventional Fmoc solid-phase peptide synthesis methods).

[0039] Step 2.2: Coating the mineral sacrificial core; 1.00 g of acid-sensitive bone mineral sacrificial nucleus was added to the above-mentioned pathogen nutrient-adhesion microdomain precursor solution and stirred at 25°C for 1 h at a stirring speed of 200 rpm to ensure that the precursor solution was fully adsorbed onto the surface of the mineral sacrificial nucleus. Subsequently, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) was added to a final concentration of 0.05% (w / v), and the solution was stirred at a wavelength of 405 nm and an intensity of 10 mW / cm². 2 Irradiation under visible light for 60 seconds caused photocrosslinking of methacrylamide gelatin on the particle surface. Hyaluronic acid-dopamine was immobilized in the crosslinked network through molecular entanglement, hydrogen bonding, and the adhesion of catechol groups, thus forming a semi-interpenetrating hydrogel thin layer containing adhesive components on the particle surface. After crosslinking, the particles were washed three times with 50 mL of phosphate buffer (pH 7.4) each time, and collected by centrifugation at 1200 rpm for 5 min to obtain intermediate particle A. Figure 2 As shown.

[0040] The pathogen nutrition-adhesion microdomain layer can provide a local adhesion environment rich in collagen-like, fibrinogen-like, and sugar-like substances, which is conducive to the adhesion and retention of bone infection-related pathogens in the subsequent restricted pathogen colonization layer within the local microdomain of the particles.

[0041] III. Construction of a restricted pathogen colonization layer: Step 3.1: Prepare the planting layer substrate solution; Take 0.30 g of sodium alginate, 0.20 g of carboxymethyl chitosan, 0.20 g of oxidized dextran, and 0.20 g of gelatin, and add them to 100 mL of phosphate buffer solution at pH 7.4. Dissolve the solution by stirring at 300 rpm for 1–2 hours at 25–37 °C to obtain the colonization layer substrate solution.

[0042] Step 3.2: Add bone infection-related pathogens; The pathogenic microorganism associated with the bone infection is Staphylococcus aureus, specifically the standard strain ATCC 25923 or MRSA USA300 in this embodiment. Staphylococcus aureus in the logarithmic growth phase was washed twice with sterile phosphate buffer (pH 7.4) and resuspended, adjusting the bacterial suspension concentration to 1 × 10⁻⁶. 8 CFU / mL.

[0043] In a biosafety cabinet, the above bacterial suspension is added to the colonization substrate solution obtained in step 3.1 at 8% (v / v) of the volume of the colonization substrate solution. The mixture is shaken and mixed on a shaker at 25°C and 150 rpm for 30 minutes to ensure that the pathogenic microorganisms are evenly dispersed in the colonization substrate solution, thus obtaining the colonization substrate solution containing bacteria.

[0044] Step 3.3: Coating intermediate particle A; In a biosafety cabinet, 1.00 g of intermediate particle A was added to a bacterial colonization layer matrix solution and gently mixed on a shaker at 25°C and 150 rpm for 30 minutes to coat the surface of intermediate particle A with the bacterial colonization layer matrix solution. Subsequently, sterile calcium chloride solution was added dropwise to induce calcium ion-induced ionic cross-linking of sodium alginate; simultaneously, the aldehyde groups in oxidized dextran reacted with the amino groups in carboxymethyl chitosan and gelatin to undergo a dynamic Schiff base reaction, thereby forming a restricted pathogen colonization layer containing pathogenic microorganisms on the particle surface. The concentration, amount, and cross-linking time of the calcium chloride solution were optimized to form a stable gel network without significantly reducing the survival rate of pathogenic microorganisms. After the reaction was complete, the particles were gently washed with sterile phosphate buffer filtered through a 0.22 μm filter membrane to remove unbound free components, yielding intermediate particle B, as shown below. Figure 3 As shown.

[0045] The confined pathogen colonization layer, through the combined action of ion cross-linking networks, dynamic covalent networks, and adhesion microdomains, restricts bone infection-related pathogens to local gel microcavities on the particle surface. This structure increases the retention probability of pathogens in the local particle area and bone defect region, and reduces the risk of their rapid diffusion out of the bone defect region in a free state.

[0046] IV. Construction of the biomimetic biological membrane ion condensation layer: Step 4.1: Prepare polyanionic solution; Take 0.30 g of sodium alginate, 0.20 g of polyglutamic acid, 0.10 g of chondroitin sulfate and 0.05 g of sodium deoxyribonucleic acid, add them to 100 mL of HEPES buffer, adjust the pH to 7.2-7.4, stir and dissolve at room temperature at 500 rpm for 2 h to obtain a polyanionic solution.

[0047] Step 4.2: Prepare a cation-bridged solution; Take 0.10 g of poly-L-lysine hydrobromide, 0.10 g of calcium chloride and 0.05 g of magnesium chloride, add them to 100 mL of HEPES buffer, adjust the pH to 7.2-7.4, and stir for 30 min at room temperature to obtain a cation-bridged solution.

[0048] Step 4.3: Formation of a biomimetic biological membrane ion condensation layer; Take 1.00 g of intermediate particle B and add it to the polyanion solution. Stir gently for 30 min at 25°C at a stirring speed of 100–200 rpm to allow the polyanion component to adsorb onto the surface of the restricted pathogen colonization layer. Then, slowly add the cation bridging solution to the above system while maintaining gentle stirring. After the addition is complete, continue the reaction for 1 h to obtain the biomimetic biomembrane ion-condensed layer. After the reaction is complete, gently wash with sterile HEPES buffer, collect the particles, and obtain intermediate particle C, as shown below. Figure 4 As shown.

[0049] This biomimetic biomembrane ion condensation layer contains polysaccharides, polyanions, nucleic acid-like components, and a metal ion bridging network. It can simulate part of the composition and barrier characteristics of the extracellular polymer matrix of bacterial biomembranes, improve the viscoelasticity of the local gel network of particles, and reduce the diffusion rate of macromolecular components, thereby providing a supporting environment for bone infection-related pathogens to form biomembrane-like aggregation structures locally on particles.

[0050] V. Construction of the Immune Metabolic Regulatory Layer: Step 5.1: Preparation of lactate-succinate sustained-release microparticles; 0.50 g of polylactic acid-glycolic acid copolymer was added to 20 mL of dichloromethane and stirred at room temperature at 400 rpm for 30 min to obtain the organic phase. 0.20 g of sodium lactate and 0.10 g of sodium succinate were added to 2 mL of deionized water and stirred until dissolved. This dissolved mixture was then added to the organic phase and emulsified using a high-speed shear method to form a primary emulsion. The primary emulsion was added to 200 mL of a 1.0% polyvinyl alcohol aqueous solution and emulsified by stirring at 800 rpm for 4 h, allowing the dichloromethane to evaporate and forming lactate-succinate sustained-release microparticles. After emulsification, the microparticles were centrifuged at 8000 rpm for 10 min, washed three times with 50 mL of deionized water each time, and freeze-dried for 24 h to obtain the lactate-succinate sustained-release microparticles.

[0051] Step 5.2: Prepare the immunometabolism reprogramming coating solution; Take 0.30 g of carboxymethyl chitosan and add it to 100 mL of phosphate buffer (pH 6.0). Dissolve the chitosan by stirring at 500 rpm for 2 hours at room temperature. Separately, take 0.20 g of oxidized dextran and add it to 50 mL of phosphate buffer (pH 7.4). Dissolve the oxidized dextran by stirring at 400 rpm for 1 hour at room temperature. Mix the carboxymethyl chitosan solution and the oxidized dextran solution, add 0.20 g of lactate-succinate sustained-release microparticles, adjust the pH to 7.0–7.2, and stir for 30 minutes to obtain the immunometabolism reprogramming coating solution.

[0052] Step 5.3: Coating intermediate particle C; Take 1.00 g of intermediate particle C and add it to the immunometabolism reprogramming coating solution. Gently stir the mixture for 2–3 hours under suitable conditions at a stirring speed of 100–200 rpm. During the reaction, a dynamic Schiff base network forms between carboxymethyl chitosan and oxidized dextran, immobilizing the lactate-succinate sustained-release microparticles on the outer layer of the particles, forming an immunometabolism regulatory layer. After the reaction is complete, gently wash with sterile phosphate buffer to obtain intermediate particle D, as shown below. Figure 5 As shown.

[0053] The immunometabolic regulation layer can release metabolically relevant components such as lactate and succinate at the site of bone defect, forming a chronic inflammatory-like metabolic microenvironment. This layer, in conjunction with the restricted pathogen colonization layer and the biomimetic biomembrane ion aggregation layer, helps prolong the duration of local inflammatory stimulation and improves the stability of the infectious inflammatory response in the model.

[0054] VI. Construction of the osteoclast activation regulatory layer: Step 6.1: Prepare the lipid film; 0.20 g of hydrogenated soybean lecithin, 0.05 g of cholesterol, and 0.03 g of DSPE-PEG-COOH were added to a chloroform / methanol mixed solvent, with 20 mL of chloroform and 10 mL of methanol. After stirring and dissolving at room temperature for 30 min, the solution was placed in a rotary evaporator and evaporated under reduced pressure at 40 °C for 30 min to form a uniform lipid film. The lipid film was then dried in a vacuum desiccator for 2 h to remove residual organic solvent.

[0055] Step 6.2: Hydration to form biomimetic exosome vesicles; 50 mL of phosphate buffer (pH 7.4) was added to the lipid membrane, and the mixture was hydrated at 45 °C for 1 h with stirring at 300 rpm. The resulting lipid suspension was then gently homogenized to form an exosome-inspired vesicle suspension.

[0056] Step 6.3: Couple bone-targeting peptides and osteoclast-inducing mimic peptides; EDC 20 mg and NHS 12 mg were added to the exosome-inspired vesicle suspension, and the pH was adjusted to 5.8–6.0. ​​The mixture was reacted at room temperature for 30 min to activate the carboxyl groups on DSPE-PEG-COOH. Subsequently, 10 mg of bone-targeting peptide and 10 mg of osteoclast-inducing mimic peptide were added, and the pH was adjusted to 7.2–7.4. The mixture was reacted at room temperature for 4 h with a stirring speed of 200 rpm. After the reaction was completed, the system was placed in a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed against phosphate buffer at pH 7.4 for 12 h, with the buffer being changed every 4 h to remove free peptides and small molecule activators. The resulting product was an osteoclast-inducing exosome-inspired vesicle suspension.

[0057] The bone-targeting peptide is an octapartic acid peptide, specifically NH2-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp in this embodiment. The bone-targeting peptide, through the affinity of its multiple aspartic acid residue side chain carboxyl groups for calcium ions, hydroxyapatite, or calcium phosphate structures on the surface of bone minerals, thereby endowing exosomes with biomimetic vesicle bone-targeting capabilities.

[0058] The osteoclast-inducible mimic peptide is a fragment of human parathyroid hormone 1-34, i.e., hPTH(1-34). Under continuous local stimulation, this fragment can indirectly participate in osteoclast activation-related processes by influencing osteocyte, osteoblast, or bone marrow microenvironment-related signals.

[0059] Step 6.4: Embed the exosome-like biomimetic vesicles into the outer layer of the particles; Take 1.00 g of intermediate particle D and add it to 50 mL of osteoclast-inducing exosome biomimetic vesicle suspension. Incubate gently at 37°C and 100 rpm for 2 hours. During this process, the exosome biomimetic vesicles are immobilized in the outer network of intermediate particle D through various non-covalent interactions, such as the affinity between bone-targeting peptides and calcium-phosphorus mineral phases, hydrophobic interactions between lipid membranes and hydrophobic regions in the polymer network, and hydrogen bonding and electrostatic interactions, forming an osteoclast-activating regulatory layer. After the reaction is complete, gently wash with sterile phosphate buffer to obtain intermediate particle E, as shown below. Figure 6 As shown.

[0060] The osteoclast activation regulatory layer can gradually release or expose bone resorption regulatory signals in the infectious inflammatory microenvironment, linking the inflammatory response caused by pathogen colonization with the local bone resorption process, thereby improving the consistency of bone destruction phenotypes in animal models of infectious bone defects.

[0061] VII. Construction of a hypoxia-acidification microenvironment conditioning layer: Step 7.1: Prepare the coating solution for the low-oxygen acidification microenvironment conditioning layer; Take 0.20 g of poly-L-histidine hydrochloride and add it to 50 mL of MES buffer. Adjust the pH to 6.5 and stir at room temperature for 1 h at a stirring speed of 400 rpm. Add 0.05 g of glucose oxidase, 0.05 g of MIL-100(Fe) nanoparticles, 0.05 g of branched polyethyleneimine, and 0.10 g of gluconolactone. Continue stirring for 30 min at a stirring speed of 300 rpm to obtain the hypoxia-acidified microenvironment conditioning layer coating solution.

[0062] Step 7.2: Coat the intermediate particle E; Take 1.00 g of intermediate particle E and add it to the above-mentioned hypoxia-acidification microenvironment conditioning layer coating solution. Stir at 150 rpm for 2 hours at room temperature. During this process, electrostatic interactions occur between branched polyethyleneimine, poly-L-histidine, and anionic groups such as carboxyl, sulfate, and phosphate groups in the outer layer of the particle. Simultaneously, the pH responsiveness, hydrogen bonding, and hydrophobic interactions of poly-L-histidine jointly promote the deposition of these components on the surface of intermediate particle E, forming a hypoxia-acidification microenvironment conditioning layer. Components such as glucose oxidase, MIL-100(Fe) nanoparticles, and gluconolactone in the system are co-deposited or co-embedded within this deposition layer, forming a hypoxia-acidification microenvironment conditioning layer. After the reaction is complete, gently wash with sterile phosphate buffer to obtain intermediate particle F, as shown below. Figure 7 As shown.

[0063] This hypoxic-acidified microenvironment conditioning layer can promote oxygen consumption, gluconic acid production, and oxidative stress-related responses in the presence of local tissue fluid and nutrient substrates, thereby forming a local microenvironment around the particles where hypoxia, acidification, and oxidative stress coexist. This microenvironment is beneficial for mimicking the metabolic imbalance state in infectious bone defect lesions and coordinates with pathogen colonization, biofilm-like structure formation, and bone mineral dissolution processes.

[0064] VIII. Construction of the anti-escape confinement hydrogel shell: Step 8.1: Prepare the shell precursor fluid; Take 0.40 g of sulfobetaine methacrylate, 0.30 g of β-cyclodextrin-modified hyaluronic acid, 0.20 g of adamantane-modified gelatin, and 0.10 g of methoxy polyethylene glycol phosphonic acid, and add them to 100 mL of phosphate buffer solution at pH 7.4. Stir and dissolve at 25 °C for 2 h at a stirring speed of 400 rpm to obtain the shell precursor solution.

[0065] Among them, sulfobetaine methacrylate is used to form a double-charged hydration network; β-cyclodextrin-modified hyaluronic acid and adamantane-modified gelatin form a reversible self-healing network through host-guest interaction; and phosphonic acid-modified polyethylene glycol is used to enhance the affinity between particles and bone mineral interfaces.

[0066] The preparation method of the adamantane-modified gelatin is as follows: Take 1.00 g of gelatin and add it to 100 mL of 0.05 mol / L MES buffer. Adjust the pH to 5.5–6.0 and stir to dissolve at 40 °C for 1–2 h to obtain a gelatin solution. Separately, take 0.20 g of 1-adamantane carboxylic acid and dissolve it in 10 mL of anhydrous dimethyl sulfoxide. Then add 0.32 g of EDC·HCl and 0.19 g of NHS and stir to activate at room temperature for 30–60 min to obtain an activated 1-adamantane carboxylic acid solution. Slowly add the activated solution dropwise to the gelatin solution and react at 35–40 °C and pH 5.5–6.2 for 12–24 h to allow the amino groups on the gelatin molecular chain to undergo an amidation reaction with the carboxyl groups of 1-adamantane carboxylic acid. After the reaction was completed, the resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da. Dialysis was first performed with deionized water or phosphate buffer at pH 7.4 for 12 hours, followed by dialysis with deionized water for 48-72 hours, with the dialysis solution changed every 6-8 hours. After dialysis, the product was pre-frozen at -20°C to -80°C for 4-12 hours and then freeze-dried for 24-48 hours to obtain adamantane carboxylic acid grafted gelatin.

[0067] Step 8.2: Add crosslinking agent; Add 0.01 g of N,N'-methylenebisacrylamide to the shell precursor fluid and stir for 10 min to ensure complete dispersion. Then add 0.01 g of ammonium persulfate and continue stirring for 5 min.

[0068] Step 8.3: Shell coating; Take 1.00 g of intermediate particle F and add it to the above-mentioned shell precursor solution. Stir gently for 20 min at 100–150 rpm under sterile conditions (25℃, pH 7.4) to ensure the precursor solution fully wets and adsorbs onto the outer layer of the particle. Then, add 20 μL of N,N,N',N'-tetramethylethylenediamine and react at 25℃ for 30 min to induce free radical cross-linking of sulfobetaine methacrylate, which then forms an escape-preventing confined hydrogel shell with the β-cyclodextrin-adamantane host-guest network. After the reaction is complete, gently wash with sterile phosphate buffer and collect the particles to obtain wet pathogen-confined infectious bone defect modeling microreactor particles. The wet-state pathogen-confined infectious bone defect modeling microreactor particles were dispersed in sterile phosphate buffer containing lyophilization protectants, namely trehalose and mannitol, and pre-frozen at -20°C to -80°C for 4–12 h, followed by freeze-drying for 24–48 h to obtain dry-state microreactor particles. Figure 8 As shown.

[0069] This anti-escape confinement hydrogel shell, as an outer spatial barrier structure, can reduce the risk of pathogenic microorganisms, inflammatory stimuli and functional particles rapidly spreading out of the bone defect area, while allowing small molecule nutrients, metabolites and tissue fluid to exchange to a certain extent, thus taking into account both local confinement and microenvironment response.

[0070] Example 2: The pathogen-confined infectious bone defect modeling microreactor particles are further dispersed in an injectable externally sealed hydrogel carrier to form a dual-confined structure.

[0071] Step 1: Prepare a catechin-modified gelatin solution; Take 4.00 g of catechin-modified gelatin and add it to 80 mL of sterile phosphate buffer at pH 7.4. Stir and dissolve the gelatin at 37°C for 1 hour at a stirring speed of 300 rpm to obtain a 5% (w / w) catechin-modified gelatin solution.

[0072] The method for preparing the catechin-modified gelatin is as follows: 1.00 g of gelatin is added to 100 mL of 0.05 mol / L MES buffer, the pH is adjusted to 5.5–6.0, and the mixture is stirred and dissolved at 40°C for 1–2 h to obtain a gelatin solution. 0.32 g of EDC·HCl and 0.19 g of NHS are added to the gelatin solution, and the mixture is reacted at room temperature for 15–30 min to activate the carboxyl groups on the gelatin molecular chain. Separately, 0.20 g of dopamine hydrochloride is dissolved in 10 mL of 0.05 mol / L MES buffer to obtain a dopamine solution. The dopamine solution is slowly added dropwise to the EDC / NHS-activated gelatin solution, and the mixture is reacted in the dark at 35–40°C and pH 5.5–6.2 for 12–24 h to allow the amino groups in the dopamine molecule to undergo an amidation reaction with the activated carboxyl groups on the gelatin molecular chain. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da and dialyzed with deionized water for 48-72 hours, with the dialysate being replaced every 6-8 hours. After dialysis, the gelatin was pre-frozen at low temperature and freeze-dried to obtain catechol-modified gelatin.

[0073] Step 2: Prepare an oxidized hyaluronic acid solution; Take 2.00 g of hyaluronic acid-aldehyde (HA-CHO) and add it to 20 mL of sterile phosphate buffer at pH 7.4. Stir and dissolve at room temperature for 2 h at a stirring speed of 300 rpm to obtain an oxidized hyaluronic acid solution with a mass concentration of 10%.

[0074] Step 3: Disperse the microreactor particles; Add the microreactor particles for pathogen-limited infectious bone defect modeling to a catechol-modified gelatin solution, and gently mix under sterile, low-shear conditions at 4–25°C to ensure uniform dispersion of the microreactor particles in the solution. (Preferred mixing methods include slow pipetting 5–10 times, repeated injection with a double syringe 5–15 times, or low-speed stirring at 50–150 rpm for 1–5 minutes).

[0075] Step 4: Formation of a particle-gel composite system; An oxidized hyaluronic acid solution was added to a catechol-modified gelatin solution containing microreactor particles, maintaining a volume ratio of 4:1 between the catechol-modified gelatin solution and the aldehyde-modified sodium hyaluronate solution. The two solutions were rapidly mixed by reciprocating injection 5–15 times using a dual-syringe connector or by low-speed vortexing for 5–15 seconds. This process induces a dynamic Schiff base reaction between the aldehyde groups in the oxidized hyaluronic acid and the amino groups in the catechol-modified gelatin, forming an injectable, externally sealed hydrogel carrier.

[0076] The resulting particle-gel composite system can be formed in situ within the bone defect cavity, confining multiple microreactor particles to the local area of ​​the bone defect, thereby forming a dual local infection control system with intraparticle confinement and hydrogel confinement.

[0077] Usage: The pathogen-restricted infectious bone defect modeling microreactor particles of the present invention are used to construct animal models of infectious bone defects.

[0078] In one embodiment, a standardized bone defect cavity is first formed in the long bone region of a healthy experimental animal, and then the particle-gel composite system of the present invention is placed into the bone defect cavity. After implantation, the restricted pathogen colonization layer in the microreactor particles enables bone infection-related pathogenic microorganisms to colonize locally in the bone defect; the pathogen nutrition-adhesion microdomain layer provides a local adhesion environment for pathogenic microorganisms; the biomimetic biomembrane ion aggregation layer promotes the formation of local biomembrane-like structures; the immune metabolism regulation layer and the hypoxia acidification microenvironment regulation layer maintain the infectious inflammatory microenvironment; the osteoclast activation regulation layer promotes the synchronous occurrence of infectious inflammation and bone resorption processes; the acid-sensitive bone mineral sacrificial core undergoes dissolution in the acidic inflammatory environment; and the anti-escape confinement hydrogel shell and the external sealing hydrogel carrier together restrict the diffusion of pathogenic microorganisms and inflammatory stimuli out of the bone defect area.

[0079] As a result, the pre-fabricated mechanical bone defect area gradually transforms into an infectious bone defect-like lesion characterized by pathogen colonization, biofilm formation, inflammatory infiltration, and bone tissue destruction.

[0080] Example 3: In vitro verification of pathogen confined colonization and biomimetic biofilm formation effects.

[0081] This embodiment is used to verify the synergistic effect of the restricted pathogen colonization layer and the biomimetic biofilm ion condensation layer, that is, whether pathogenic microorganisms are effectively confined to the local part of the particle and form a biofilm-like structure on the particle surface.

[0082] I. Experimental Groups: Experimental group: The intact microreactor particles containing Staphylococcus aureus prepared in Example 1 had a bacterial count of approximately 1 × 10⁻⁶ per milligram of particle. 5 CFU.

[0083] Control group 1: Equal volume of free Staphylococcus aureus suspension.

[0084] Control group 2: Simplified microreactor particles lacking the biomimetic biomembrane ion condensation layer. The preparation method is the same as in Example 1, but the fourth part "Construction of biomimetic biomembrane ion condensation layer" is omitted. The remaining coating steps are kept the same, and the bacterial content is comparable to that of the experimental group.

[0085] II. Bacterial Leakage Detection: Each group of samples was placed in a sterile centrifuge tube containing 5 mL of tryptone soy broth and incubated with shaking at 37°C and 100 rpm. Samples were taken at 24 h, 48 h, and 72 h after incubation, and 100 μL of the supernatant was taken, serially diluted, and spread onto tryptone soy agar plates. After incubation at 37°C for 24 h, colony counting was performed.

[0086] Depend on Figure 9 It is evident that the bacterial count in the supernatant of the free Staphylococcus aureus group remained at a high level throughout the culture process, and further increased with prolonged culture time. At 24 hours, the bacterial count in the supernatant reached approximately 7.15 log₂O₅. 10 CFU / mL increased to approximately 8.76 log⁻¹ by 72 h. 10 CFU / mL. This indicates that free bacteria lack spatial confinement structures, allowing them to rapidly proliferate and spread in liquid culture environments, making it difficult to maintain local colonization. The simplified microreactor particle group lacking the biomimetic biomembrane ion condensation layer showed lower supernatant bacterial leakage than the free bacteria group, but it still increased significantly over time. The supernatant leakage bacterial counts in this group at 24h, 48h, and 72h were approximately 4.18, 5.02, and 5.64 log₂O₅ / mL, respectively. 10 The CFU / mL level suggests that while a restricted pathogen colonization layer can reduce bacterial escape to some extent, the lack of a polysaccharide, polyanion, and metal ion bridging barrier provided by a biomimetic biomembrane ion condensation layer allows bacteria and related components to gradually diffuse into the external culture environment. In contrast, the intact microreactor particle group exhibited the lowest bacterial leakage in its supernatant, with only approximately 2.35 log [value missing] at 24 h. 10 CFU / mL, increased to approximately 3.06 log at 72 h. 10The CFU / mL increase was relatively small overall. This indicates that the intact microreactor particles effectively limit the diffusion of Staphylococcus aureus into the external environment, keeping the pathogenic microorganisms mainly confined to the local area of ​​the particle. This result demonstrates that the locally confined structure formed by the restricted pathogen colonization layer and the biomimetic biomembrane ion condensation layer can significantly reduce the risk of bacterial leakage, which is beneficial for simulating the local colonization state of pathogenic microorganisms in infectious bone defect lesions.

[0087] Example 4: Verification of the dissolution behavior of acid-sensitive bone mineral sacrificial cores.

[0088] This embodiment is used to verify the preferential dissolution behavior of acid-sensitive bone mineral sacrificial nuclei in an acidic infected microenvironment.

[0089] I. pH-dependent dissolution experiment: 50 mg of the acid-sensitive bone mineral sacrificial core powder prepared in Example 1 was soaked in 50 mL of phosphate buffer solution at pH 7.4 (simulating physiological environment) and pH 5.5 (simulating acidified infection microenvironment), respectively, and incubated in a shaker at 37°C and 100 rpm. 5 mL samples were taken on days 1, 3, 7, 14, and 21 after incubation, with an equal volume of buffer solution at the corresponding pH added after each sampling. The samples were filtered through a 0.22 μm filter membrane, and the Ca2+ content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+ Mg 2+ 、Sr 2+ The cumulative release amount.

[0090] according to Figures 10-12 The results showed that acid-sensitive bone mineral sacrificial nuclei exhibited significant differences under different pH conditions. Compared with the physiological environment of pH 7.4, the acidified environment of pH 5.5 significantly promoted the formation of calcium-sensitive bone mineral sacrificial nuclei. 2+ Mg 2+ and Sr 2+ The release of the minerals significantly increased the overall relative mineral dissolution rate of the material, indicating that the bone mineral sacrificial core has a clear pH-dependent dissolution characteristic.

[0091] 1.Ca 2+ Cumulative release results analysis: Depend on Figure 10 It can be seen that under pH 7.4 conditions, Ca 2+ The cumulative release concentration increased slowly with prolonged incubation time. It was approximately 6.2 mg / L on day 1, rising to approximately 17.6 mg / L on day 7, and reaching approximately 31.5 mg / L on day 21. This result indicates that in an environment close to physiological pH, acid-sensitive bone mineral sacrificial nuclei undergo only mild and slow mineral ion release, with a relatively stable overall structure. In contrast, under pH 5.5 conditions, Ca... 2+ Release accelerated significantly. On day 1, Ca... 2+The cumulative release concentration reached approximately 28.4 mg / L, higher than the level in the pH 7.4 group during the same period; it rose to approximately 122.6 mg / L on day 7, approximately 178.3 mg / L on day 14, and further increased to approximately 212.5 mg / L on day 21. This indicates that an acidic environment can significantly promote the dissolution of calcium components in the bone mineral phase. These results are consistent with the acid-sensitive characteristics of strontium / magnesium co-doped carbonated amorphous calcium phosphate materials. Under acidic conditions, the calcium phosphate mineral phase is more prone to proton-induced dissolution, leading to the release of Ca... 2+ Continuous release. Therefore, Ca 2+ The release results can directly reflect the mineral destruction process of the sacrificial nucleus in the infected acidified microenvironment.

[0092] 2.Mg 2+ Cumulative release results analysis: Depend on Figure 11 It can be seen that Mg 2+ Release trend and Ca 2+ The results were largely consistent, but the overall release concentration was lower than that of Ca. 2+ This is consistent with its low doping content in the sacrificial nucleus. At pH 7.4, Mg... 2+ The release was relatively slow, approximately 0.18 mg / L on day 1, approximately 0.51 mg / L on day 7, and only rose to approximately 0.91 mg / L on day 21. This indicates that under neutral or weakly alkaline physiological conditions, the magnesium-doped mineral phase did not undergo significant rapid dissolution. At pH 5.5, Mg... 2+ Release was significantly enhanced. It was approximately 1.15 mg / L on day 1, rising to approximately 4.15 mg / L on day 7, and reaching approximately 6.36 mg / L on day 21. Compared to the pH 7.4 group, the pH 5.5 group showed higher Mg levels at all time points. 2+ The release levels indicate that acidic environments not only promote the dissolution of the calcium phase but also simultaneously promote the release of magnesium dopant from the mineral structure. This result suggests that magnesium is not simply adsorbed onto the particle surface but participates in the doping structure of the bone mineral sacrificial nucleus; when an acidic environment leads to the dissolution of the mineral phase, Mg... 2+ This release indirectly supports the destruction of the overall mineral structure of the sacrificial nucleus, rather than a single surface ion exchange.

[0093] 3.Sr 2+ Cumulative release results analysis: Depend on Figure 12 It can be seen that Sr 2+ It also exhibits a clear pH-dependent release behavior.

[0094] Under pH 7.4 conditions, Sr 2+The cumulative release concentration was low, approximately 0.21 mg / L on day 1, approximately 0.66 mg / L on day 7, and approximately 1.22 mg / L on day 21. This indicates that the strontium component is released slowly under physiological conditions, and the material structure possesses a certain degree of stability. Under pH 5.5 conditions, Sr... 2+ Release increased significantly. It was approximately 1.42 mg / L on day 1, rising to approximately 5.34 mg / L on day 7, reaching approximately 7.15 mg / L on day 14, and further increasing to approximately 8.08 mg / L on day 21. This indicates that acidic conditions can promote the dissolution of strontium-doped mineral structures, allowing Sr to release more minerals. 2+ Continuous release. Sr 2+ Release trend and Ca 2+ and Mg 2+ The consistency indicates that the acid-sensitive bone mineral sacrificial core undergoes a multi-ion coordinated release process in the infected acidified environment. This phenomenon is consistent with the material characteristics of strontium / magnesium co-doped carbonated amorphous calcium phosphate undergoing overall dissolution under acidic conditions.

[0095] Example 5: Verification of the effect of animal modeling of infectious bone defects.

[0096] This embodiment is used to verify the effect of the microreactor particles of the present invention in constructing infectious bone defect-like lesions in experimental animals.

[0097] I. Laboratory Animals and Modeling: like Figures 13-15 Twenty-seven male SPF-grade SD rats (weighing 250–300 g) were selected and approved by the ethics committee. They were randomly divided into three groups of nine rats each.

[0098] Blank control group: A standardized bone defect with a diameter of 2 mm and a depth of 5 mm was prepared in the femoral condyle, without implanting any material.

[0099] Traditional inoculation group: After preparing the same bone defect, Staphylococcus aureus (approximately 1×10⁻⁶) was injected. 6 20 μL of physiological saline suspension of CFU.

[0100] Microreactor Particle Assembly: After preparing identical bone defects, a microreactor particle-gel composite system containing an equal amount of bacteria (approximately 10 particles, total bacterial count approximately 1×10⁻⁶) was implanted according to the method in Example 2. 6 CFU).

[0101] The surgery was performed under general anesthesia and strict aseptic conditions. Postoperatively, the patient was fed routinely and allowed free access to food and water.

[0102] II. Bacterial load detection: On postoperative days 7, 14, and 28, three rats from each group were randomly sacrificed. Under aseptic conditions, the femoral defect area and surrounding bone tissue (approximately 100 mg) were removed, weighed, homogenized, and serially diluted for plate counting.

[0103] Depend on Figure 16 As can be seen, the bacterial load in the blank control group remained at a very low level on postoperative days 7, 14, and 28, approximately 1.00–1.05 log. 10 The CFU / g level indicates that simple mechanical bone defects did not lead to significant infection in the absence of exogenous pathogen stimulation. The conventional inoculation group had a higher bacterial load on postoperative day 7, approximately 5.82 log [missing value]. 10 The CFU / g level indicates that direct injection of Staphylococcus aureus can induce local infection in the early stages. However, the bacterial load in this group decreased significantly over time, dropping to approximately 4.76 log on day 14. 10 CFU / g further decreased to approximately 3.48 log on day 28. 10 CFU / g. This indicates that free bacteria are easily affected by factors such as tissue fluid flushing, local immune clearance, and diffusion in vivo, making it difficult for them to remain stably in the bone defect area for a long period. The microreactor particle group maintained a high bacterial load on postoperative days 7, 14, and 28, at approximately 5.96, 5.72, and 5.38 log, respectively. 10 CFU / g. Compared with the conventional inoculation group, the bacterial load in this group decreased less, indicating that the microreactor particles can effectively limit the escape of pathogenic microorganisms and promote their continuous colonization in the bone defect area. This result shows that the microreactor particles, through the combined action of a confined pathogen colonization layer, an escape-preventing hydrogel shell, and an externally encapsulating hydrogel carrier, can improve the retention stability of pathogenic microorganisms in the bone defect area.

[0104] The in vivo experimental results above demonstrate that the microreactor particles of this invention can increase the sustained retention level of pathogenic microorganisms within the bone defect area and induce infectious bone defect-like pathological changes in the pre-fabricated mechanical bone defect area. Compared with the traditional direct inoculation method, the microreactor particle group of this invention exhibited a more stable local bacterial load during the postoperative observation period, suggesting that it is beneficial to improve the stability and reproducibility of the infectious bone defect animal model.

[0105] Working principle: The pathogen-confined infectious bone defect modeling microreactor particles of this invention are multilayer functional materials used to construct infectious bone defect-like lesions within pre-fabricated bone defect areas. Their working principle is not simply to directly add pathogenic microorganisms to the bone defect area, but rather through a continuous process of "pathogen-confined colonization—biofilm pre-organization—immunometabolic reprogramming—hypoxia-acidification self-feedback—osteoclast-induced coupling—sacrificial dissolution of bone minerals—dual anti-exodus confinement," gradually transforming ordinary mechanical bone defects into infectious bone defect lesions characterized by pathogen colonization, biofilm formation, inflammatory infiltration, bone resorption, and bone tissue destruction.

[0106] Specifically, after the microreactor particles are implanted into the prefabricated bone defect cavity, the outer anti-escape confinement hydrogel shell and the external sealing hydrogel carrier first form a spatially confined structure in the local bone defect area, causing the particles, pathogenic microorganisms, and functional components to be mainly retained within the bone defect cavity. Subsequently, the pathogen nutrition-adhesion microdomain layer provides a local adhesion environment rich in collagen-like, fibrinogen-like, sugar, and ions for bone infection-related pathogenic microorganisms, enabling them to colonize on the particle surface and in the local bone defect area.

[0107] The restricted pathogen colonization layer, through gel microcavities and dynamic cross-linked networks formed by alginate, chitosan, gelatin, and oxidized dextran, confines pathogenic microorganisms within the local space of the particles, preventing their rapid spread in a free state. This structure preserves the basis of actual pathogen colonization while improving the locality and controllability of the infected area.

[0108] After the pathogenic microorganisms colonize locally, the biomimetic biofilm ion condensation layer plays a further role. This layer is composed of polysaccharides, polyanions, nucleic acid-like polymers, cationic short peptides, and a multivalent metal ion bridging network, which can simulate the high viscoelasticity, low diffusion, and ion cross-linking characteristics of the extracellular polymer matrix of bacterial biofilms. On the one hand, this layer is conducive to the formation of biofilm-like structures by pathogenic microorganisms locally; on the other hand, it can restrict the rapid exchange of substances inside and outside the infection site, making the infection focus exhibit local barrier characteristics that are closer to chronic infection.

[0109] Metabolic regulatory components such as lactate and succinate in the immunomodulatory layer are gradually released, creating a metabolically disordered microenvironment at the site of bone defect, keeping local immune cells in a state of persistent inflammatory response. This layer works synergistically with the pathogen colonization layer and biofilm-like structures to maintain the infectious inflammatory response, rather than just producing transient acute stimulation.

[0110] The glucose oxidase-like nanozymes, gluconolactones, pH-responsive polymers, and metal-organic coordination nanoparticles in the hypoxic-acidified microenvironment regulation layer further modulate local oxygen tension, acid-base status, and oxidative stress levels, creating a coupled state of hypoxia, acidification, and reactive oxygen species in the bone defect area. This state is similar to the metabolic microenvironment in infectious bone defect-like lesions, which can further promote pathogen colonization, biofilm stability, and persistent inflammation.

[0111] The exosome-inspired vesicles, bone-targeting peptides, and osteoclast-inducing simulated signals in the osteoclast activation regulatory layer are used to simulate intercellular vesicle communication and osteoclast activation processes under infectious inflammatory conditions. This layer enables synchronous coupling between infectious inflammation and osteoclast activation, bone resorption, and bone defect expansion, thereby avoiding the problems of "infection formation but insufficient bone destruction" or "mechanical defects present but unstable infectious pathology" in traditional models.

[0112] The acid-sensitive bone mineral sacrificial core is located in the inner layer of the granules and is composed of carbonated amorphous calcium phosphate doped with elements such as strontium and magnesium. This type of mineral structure is more susceptible to dissolution in acidic inflammatory environments than ordinary crystalline hydroxyapatite. As the acidification of the local infection focus increases, the mineral sacrificial core gradually dissolves, releasing ions such as calcium, phosphorus, magnesium, and strontium, simulating the process by which the bone mineral matrix is ​​destroyed by inflammation and the acidic microenvironment in infected bone defects.

[0113] Therefore, the material described in this invention enables pathogen colonization, inflammation maintenance, biofilm formation, hypoxia acidification, osteoclast activation, and bone mineral destruction to occur synergistically in the same local space through the continuous response of the multilayer functional structure, thereby constructing an animal model of infectious bone defects.

[0114] The workflow of the material of this invention after implantation into the prefabricated bone defect cavity can be divided into the following stages: Phase 1: Localized Detention and Spatial Confinement Phase; After the material enters the prefabricated bone defect cavity, the anti-escape confinement hydrogel shell first undergoes hydration and swelling, and then comes into contact with the tissue fluid within the bone defect cavity. If the microreactor particles are further used in conjunction with an external injectable sealing hydrogel carrier, the external hydrogel carrier can be formed in situ within the bone defect cavity, fixing multiple microreactor particles within the bone defect area.

[0115] At this stage, the material forms a two-level confinement structure: an escape-preventing confinement hydrogel shell within the microreactor particles themselves; and an injectable, sealing hydrogel carrier encapsulating the particles. This dual confinement structure concentrates pathogens, functional components, and the particle itself primarily within the pre-fabricated bone defect area, thereby reducing the risk of free pathogen spread and non-targeted inflammatory responses in surrounding soft tissues.

[0116] The second stage: pathogen adhesion and restricted planting stage; After local confinement is complete, the pathogen nutrient-adhesion microdomain layer begins to function. This layer contains collagen-like components, fibrinogen-like components, hyaluronic acid derivatives, carbohydrate components, and calcium and magnesium ions, which can simulate the extracellular matrix environment suitable for pathogen adhesion in bone wounds and infection foci. Staphylococcus aureus in the confined pathogen colonization layer undergoes local adhesion and initial colonization in this microdomain. Because the pathogenic microorganisms are embedded or semi-fixed within the gel microcavities, dynamic cross-linking network, and adhesion matrix, they do not diffuse in a completely free form, but rather form local pathogen-rich areas around the microreactor particles.

[0117] The main purpose of this stage is to establish a real infection basis, so that ordinary bone defect areas have the conditions for pathogen colonization.

[0118] Third stage: Biofilm-like barrier formation stage; After pathogenic microorganisms colonize the particle surface, the biomimetic biomembrane ion condensation layer further promotes the formation of local biomembrane-like structures.

[0119] The polysaccharide, polyglutamic acid, chondroitin sulfate, nucleic acid-like polyanions, and multivalent metal ion bridging network in this layer can mimic the main structural features of the extracellular polymer matrix of bacterial biofilms. This network has high viscoelasticity and low diffusivity, and can form a local barrier environment.

[0120] This stage causes the material to gradually form a local biofilm-like structure similar to a chronic infection lesion, characterized by: enhanced retention of pathogenic microorganisms; slowed diffusion of components; improved local resistance to clearance; persistent infection stimulation; and the transformation of the inflammatory response from transient stimulation to persistent stimulation.

[0121] Phase 4: Immune metabolic reprogramming phase; As pathogens colonize and biofilm-like structures form, lactate-succinate sustained-release microparticles in the immune metabolic regulatory layer gradually release metabolic regulatory components.

[0122] Lactate, succinate, and their associated metabolic microenvironment can mimic the metabolic disorders commonly found in infectious bone defect lesions, keeping local immune cells in a state of persistent inflammatory response. This process can induce or maintain pathological processes such as upregulation of local inflammatory factors, macrophage inflammatory-like polarization, and increased expression of matrix degradation factors. The role of this stage is to establish a sustained interaction between pathogenic stimulation and the host immune response, rather than a single, transient, and uncontrollable inflammatory stimulus.

[0123] Fifth stage: Hypoxia-acidification microenvironment regulation stage; The hypoxia-acidification microenvironment regulation layer gradually activates in the presence of local tissue fluid and nutrient substrates. Glucose oxidase-like nanozymes in this layer initiate a mild oxygen-consuming process, gluconolactone and pH-responsive polymers promote local acidification, and metal-organic coordination nanoparticles regulate local oxidative stress. Thus, a hypoxia-acidification-reactive oxygen species-coupled microenvironment gradually forms around the microreactor particles.

[0124] This microenvironment has three functions: promoting the local retention of pathogenic microorganisms and biofilm stability; maintaining infectious inflammatory responses; and triggering subsequent responses of acid-sensitive bone mineral sacrificial nuclei and osteoclast-activated regulatory layers.

[0125] The hypoxia-acidification microenvironment regulation layer enables the material to not only passively bear pathogenic microorganisms, but also to continuously influence oxygen tension, acid-base status and oxidative stress level in the presence of local tissue fluid and nutrient substrates, thus more closely resembling the characteristics of the continuously changing metabolic microenvironment in the infection focus.

[0126] Stage 6: Infection-osteoclast coupling stage; Under conditions of infectious inflammation, hypoxia-acidification, and metabolic disorders, exosome-like vesicles in the osteoclast activation regulation layer gradually release or expose osteoclast-inducing simulated signals. This layer can mimic vesicle-like signaling transmission between inflammatory cells, osteocytes, osteoblasts, and osteoclast precursor cells in infected bone defects, thereby enhancing local osteoclast activity. The results include: osteoclast-like cell activation; increased RANKL / OPG ratio; increased TRAP-positive osteoclast-like cells; upregulation of bone resorption-related indicators such as Cathepsin K and MMP-9; and aggravated bone tissue destruction in the bone defect area.

[0127] This stage synchronizes the infection response with the bone resorption process, which is an important mechanism that distinguishes this invention from ordinary bacterial hydrogels or simple pathogen inoculation models.

[0128] Stage 7: Sacrificial dissolution of bone minerals; As local acidification and inflammation intensify, the acid-sensitive bone mineral sacrificial core begins to gradually dissolve. This sacrificial core is composed of carbonated amorphous calcium phosphate doped with elements such as strontium and magnesium, which exhibits higher erosion sensitivity than ordinary hydroxyapatite in acidic inflammatory environments. After the mineral sacrificial core dissolves, the internal structure of the particles gradually loosens, releasing ions such as calcium, phosphorus, magnesium, and strontium.

[0129] This process simulates the destruction of bone mineral matrix by an acidic inflammatory environment in infected bone defects, causing the prefabricated bone defect cavity to gradually exhibit changes resembling infected bone defects, such as decreased bone density, enlarged bone pores, destruction of the bone cortex, and reduction of bone trabeculae.

[0130] Overall mechanism of action: Spatial fixation is achieved by a confined hydrogel shell (and an external closed hydrogel carrier) to prevent outward escape; local real pathogen colonization is achieved by a pathogen nutrition-adhesion microdomain layer and a confined pathogen colonization layer; biomembrane-like barrier formation is achieved by a biomimetic biomembrane ion aggregation layer; chronic inflammatory-like metabolic environment is maintained by an immune metabolism regulation layer; metabolic imbalance of the infection focus is simulated by a hypoxia-acidification microenvironment regulation layer; infection and bone resorption are synchronized by an osteoclast activation regulation layer; and bone mineral destruction is simulated by an acid-sensitive bone mineral sacrificial core. Ultimately, an infectious bone defect lesion with real infection and bone destruction characteristics is formed at the site of the bone defect.

[0131] This mechanism upgrades the material of this invention from a traditional pathogen inoculation tool to a microreactor for infectious bone defect lesions, enabling continuous reconstruction of key pathological links in the occurrence and development of infectious bone defects within a local space.

[0132] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. For example, in Embodiment 1, calcium phosphate-based mineral particles containing calcium, phosphorus, magnesium, strontium, and carbonate components can be directly selected as acid-sensitive bone mineral sacrificial nuclei, preferably strontium / magnesium co-doped carbonated amorphous calcium phosphate particles; in step 3.2, the bacterial suspension is added at 5% to 10% (v / v) of the volume of the colonization layer matrix liquid; in step 6.3, the mass ratio of bone-targeting peptides and osteoclast-inducing mimic peptides is in the range of 0.5 to 2, all of which can achieve the purpose of the present invention. Furthermore, the specific values ​​of each material can be scaled up or down proportionally in actual implementation, or adjusted within a conventional range, and the conventional operations exemplified can also be flexibly adjusted according to the circumstances. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing microreactor particles for modeling infectious bone defects, characterized in that, include: S1, preparation of acid-sensitive bone mineral sacrificial cores; Calcium phosphate-based mineral particles containing calcium, phosphorus, magnesium, strontium, and carbonate components were prepared as acid-sensitive bone mineral sacrificial cores. S2, constructing a pathogen nutrition-adhesion microdomain layer; Methacrylamide gelatin, hyaluronic acid-dopamine, fibrinogen mimic peptide, collagen-binding peptide, glucose, and calcium chloride were dissolved in phosphate buffer to obtain a pathogen nutrient-adhesion microdomain precursor solution; wherein the fibrinogen mimic peptide was Cys-Gly-Pro-Arg-Pro-Pro, and the collagen-binding peptide was Thr-Lys-Lys-Thr-Leu-Arg-Thr; Acid-sensitive bone mineral sacrificial nuclei were added to the pathogen nutrient-adhesion microdomain precursor fluid, and then a photoinitiator was added. After photocrosslinking, the particles were collected to obtain intermediate particle A. S3, constructing a restricted pathogen colonization layer; Sodium alginate, carboxymethyl chitosan, oxidized dextran and gelatin were dissolved in phosphate buffer to obtain the colonization layer substrate solution; Staphylococcus aureus was evenly dispersed in the colonization layer substrate solution, intermediate particles A were added, and calcium chloride solution was added dropwise to form a restricted pathogen colonization layer containing pathogenic microorganisms. The particles were washed and collected to obtain intermediate particles B. S4, constructing a biomimetic biological membrane ion condensation layer; Sodium alginate, polyglutamic acid, chondroitin sulfate and sodium deoxyribonucleic acid were dissolved in HEPES buffer to obtain a polyanionic solution. Take poly-L-lysine hydrobromide, calcium chloride and magnesium chloride, dissolve them in HEPES buffer to obtain a cation-bridging solution; Intermediate particle B was added to a polyanion solution, followed by a cation bridging solution to form a biomimetic biomembrane ion condensation layer. The particles were washed and collected to obtain intermediate particle C. S5, constructing an immune metabolic regulation layer; Polylactic acid-glycolic acid copolymer was dissolved in dichloromethane to obtain an organic phase; sodium lactate and sodium succinate were dissolved in water to obtain an aqueous phase; the aqueous phase was added to the organic phase and emulsified to form a primary emulsion; the primary emulsion was added to a polyvinyl alcohol aqueous solution for further emulsification, allowing the dichloromethane to evaporate; the microparticles were collected, washed, and dried to obtain lactate-succinate sustained-release microparticles. Carboxymethyl chitosan and oxidized dextran were dissolved separately in phosphate buffer, mixed, and then lactate-succinate sustained-release microparticles were added. The pH was adjusted to obtain an immunometabolism reprogramming coating solution. Take intermediate particle C and add it to the immunometabolic reprogramming coating solution to form an immunometabolic regulatory layer. Wash and collect the particles to obtain intermediate particle D. S6, constructing a osteoclast activation regulation layer; Hydrogenated soybean phospholipids, cholesterol, and DSPE-PEG-COOH were dissolved in a mixed solvent of chloroform and methanol, evaporated under reduced pressure to form a lipid film, dried, and then hydrated with phosphate buffer. After homogenization, an exosome biomimetic vesicle suspension was obtained. EDC and NHS were added to the obtained exosome biomimetic vesicle suspension to adjust the pH to acidic and activate the carboxyl group. Bone-targeting peptide and osteoclast-inducing mimic peptide were added to adjust the pH to weakly alkaline. After the reaction, the suspension was dialyzed to obtain osteoclast-inducing exosome biomimetic vesicle suspension. The bone-targeting peptide was an octameric aspartic peptide, which was NH2-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp, and the osteoclast-inducing mimic peptide was a fragment of human parathyroid hormone 1-34. Intermediate particle D was added to osteoclast-inducing exosome biomimetic vesicle suspension and incubated to form an osteoclast activation regulation layer. The particles were washed and collected to obtain intermediate particle E. S7, constructing a low-oxygen acidification microenvironment regulation layer; Poly-L-histidine hydrochloride was dissolved in MES buffer, the pH was adjusted, and glucose oxidase, MIL-100(Fe) nanoparticles, branched polyethyleneimine and gluconolactone were added to obtain a hypoxia acidification microenvironment conditioning layer coating solution. Take intermediate particle E and add it to the coating solution of the hypoxia acidification microenvironment conditioning layer to form a hypoxia acidification microenvironment conditioning layer. Wash and collect the particles to obtain intermediate particle F. S8, constructing an escape-proof confined hydrogel shell; Sulfobetaine methacrylate, β-cyclodextrin-modified hyaluronic acid, adamantane-modified gelatin, and methoxy polyethylene glycol phosphonic acid were dissolved in phosphate buffer to obtain the shell precursor solution. N,N'-methylenebisacrylamide, ammonium persulfate, and intermediate particles F were added to the shell precursor fluid, followed by the addition of N,N,N',N'-tetramethylethylenediamine. After the reaction was completed, the mixture was washed to obtain wet pathogen-confined infectious bone defect modeling microreactor particles.

2. The method for preparing microreactor particles for modeling infectious bone defects according to claim 1, characterized in that, S8, after obtaining microreactor particles for modeling wet pathogen-confined infectious bone defects, also includes: S9. The wet pathogen-confined infectious bone defect modeling microreactor particles were dispersed in sterile phosphate buffer containing a lyophilization protectant, pre-frozen, and then freeze-dried to obtain dry pathogen-confined infectious bone defect modeling microreactor particles.

3. The method for preparing microreactor particles for modeling infectious bone defects according to claim 1, characterized in that, S1, Preparation of acid-sensitive bone mineral sacrificial cores, specifically including: Calcium nitrate tetrahydrate, magnesium chloride hexahydrate, and strontium nitrate were dissolved in water to obtain a calcium-magnesium-strontium mixed solution; Dissolve diammonium hydrogen phosphate, sodium bicarbonate and sodium polyaspartate in water to obtain a phosphate-carbonate solution; The resulting phosphate-carbonate solution was added dropwise to a calcium-magnesium-strontium mixed solution to adjust the pH. After the reaction, the mixture was allowed to stand for aging, and the precipitate was collected, washed, and dried to obtain acid-sensitive bone mineral sacrificial core powder.

4. The method for preparing microreactor particles for modeling infectious bone defects according to claim 1, characterized in that, In S3, the *Staphylococcus aureus* strain is in the logarithmic growth phase. After washing with sterile phosphate buffer and resuspending, the concentration of the resulting bacterial suspension is 1 × 10⁻⁶. 8 CFU / mL, and add it to the planting layer substrate solution at 5%~10% of the substrate solution volume.

5. The method for preparing microreactor particles for modeling infectious bone defects according to claim 1, characterized in that, In S6, the mass ratio of the bone-targeting peptide to the osteoclast-inducing mimic peptide is 0.5 to 2.

6. The method for preparing microreactor particles for modeling infectious bone defects according to claim 1, characterized in that, In S8, the adamantane-modified gelatin is prepared by the following method: Dissolve gelatin in MES buffer to obtain a gelatin solution; Dissolve 1-adamantane carboxylic acid in anhydrous dimethyl sulfoxide, then add EDC·HCl and NHS, and activate at room temperature to obtain an activated solution of 1-adamantane carboxylic acid. The resulting 1-adamantane carboxylic acid activated solution was added dropwise to the gelatin solution. After the reaction was completed, the solution was dialyzed and dried to obtain adamantane-modified gelatin.

7. A microreactor particle for modeling infectious bone defects, characterized in that, It is prepared using the method for preparing microreactor particles for modeling infectious bone defects as described in any one of claims 1 to 6.

8. The microreactor particles for modeling infectious bone defects according to claim 7, characterized in that, The microreactor particles used for modeling infectious bone defects can be further dispersed in an injectable externally sealed hydrogel carrier to form a particle-gel composite system. The externally sealed hydrogel carrier includes catechol-modified gelatin and oxidized hyaluronic acid. The catechol-modified gelatin is formed by the amidation reaction of the carboxyl groups on the gelatin molecular chain with the amino groups in the dopamine molecule after activation by EDC / NHS.

9. The use of the microreactor particles for modeling infectious bone defects as described in claim 7 in constructing infectious bone defect-like lesion materials.

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