An injectable gel for osteomyelitis model construction and a preparation method thereof

Abstract

The application discloses an injectable gel for osteomyelitis model construction and a preparation method thereof, and relates to the technical field of medical animal models. The preparation method comprises the following steps: preparing bone adhesion temperature-sensitive polyphosphazene, preparing oxidized pullulan, preparing pathogenic enzyme-cleavable dihydrazide peptide cross-linking agent, preparing Staphylococcus aureus reservoir type bone mineralization microcavity, and mixing the above components with a Staphylococcus aureus bacterial suspension to prepare a double-reservoir load type injectable hydrogel precursor. The hydrogel precursor is a flowable sol at 2-8 DEG C and in-situ gelatinizes at 30-37 DEG C. According to the application, the Staphylococcus aureus is divided into continuous phase starting bacteria and microcavity reservoir bacteria, and a pH-sensitive mineralization shell is combined with a pathogenic enzyme-sensitive acylhydrazone cross-linking network, so that stage bacterial release triggered by an infection microenvironment is realized, the pathological process of continuous infection and gradual aggravation of osteomyelitis can be more stably simulated, and the repeatability and pathological correlation of the mouse osteomyelitis model are improved.

Description

Technical Field

[0001] This invention relates to the field of medical animal model technology, specifically to an injectable gel for constructing an osteomyelitis model and its preparation method. Background Technology

[0002] Osteomyelitis is an inflammatory disease of bone tissue and medullary cavity caused by bacterial infection. It can be triggered by factors such as open fractures, infection of orthopedic implants, traumatic contamination, hematogenous dissemination, or postoperative infection. Staphylococcus aureus is one of the common pathogens causing osteomyelitis. This bacterium has strong tissue adhesion, immune evasion, and biofilm formation capabilities, allowing it to persist persistently in bone tissue and medullary cavity. It promotes local inflammatory responses and bone destruction through virulence factors, proteases, inflammatory inducing factors, and metabolites. Therefore, establishing stable, reproducible animal models that can well simulate the pathological process of clinical osteomyelitis is of great significance for studying the pathogenesis of osteomyelitis, evaluating anti-infective drugs, screening bone repair materials, and developing treatment strategies for infected bone defects.

[0003] Currently, experimental osteomyelitis animal models are typically constructed by directly injecting a Staphylococcus aureus bacterial suspension into the bone marrow cavity, bone defect area, or around the implant. While this simple bacterial suspension injection method is relatively straightforward and can induce local infection and bone tissue inflammation to some extent, it still has significant limitations. First, the bacterial suspension lacks effective confinement within the bone marrow cavity, making it prone to leakage, diffusion, or dilution by local body fluids after injection, resulting in an unstable amount of bacteria actually remaining at the target site. Second, the uneven distribution of the bacterial suspension within the local space affects model consistency and experimental reproducibility. Third, direct injection of the bacterial suspension usually results in a single release, making it difficult to simulate the continuous bacterial colonization, gradual deterioration of the local microenvironment, and prolonged infection progression seen in clinical osteomyelitis. Especially in small animals such as mice, where the bone marrow cavity space is limited, the injection volume and local pressure are restricted, making conventional bacterial suspension modeling more prone to problems such as leakage, insufficient local infection, or unstable infection progression.

[0004] To improve local delivery and retention, studies have explored using hydrogels, sponges, gelatinous materials, alginate materials, or other biomaterials as carriers of bacteria, drugs, or inflammatory factors for local release or infection model construction. Hydrogels, due to their high water content, good tissue compatibility, and high injectability, can, to some extent, increase the residence time of the load in local tissues. Among them, thermosensitive hydrogels can remain fluid at low temperatures and undergo a sol-gel transition at body temperature, making them suitable for in-situ gelation after injection into irregular bone cavities. These materials offer certain advantages over conventional liquid delivery methods; however, existing thermosensitive hydrogels primarily serve simple embedding and space-filling functions, lacking sufficient responsiveness to the infection microenvironment and making it difficult to achieve phased release and dynamic regulation based on the development of osteomyelitis.

[0005] Furthermore, existing stimulus-responsive materials mostly focus on single mechanisms such as pH response, redox response, enzyme response, or temperature response. However, the release behavior of single pH-responsive materials is easily affected by factors such as common inflammatory acidification, local metabolic changes, or tissue hypoxia, and their correlation with the specific pathogen infection process is limited. Single enzyme-responsive materials may be affected by host-derived proteases or other non-target enzymes in complex body fluids and inflammatory tissue environments, resulting in insufficient pathogen relevance in the release process. Although simple calcium-phosphate carriers have some bone relevance, their release behavior often mainly relies on acidic dissolution and lacks a release mechanism coupled with the pathogen colonization process. Therefore, how to construct a local delivery system that can simultaneously respond to the acidic microenvironment of osteomyelitis and Staphylococcus aureus-related pathogen signals is one of the key issues in improving the stability and pathological relevance of osteomyelitis models.

[0006] On the other hand, most existing bacterial-carrying hydrogel systems employ the method of uniformly mixing bacteria into the hydrogel matrix. While this method can improve local bacterial retention, its release pattern remains relatively simple: if the hydrogel network is too loose, bacteria may be released in large quantities in the early stages, resulting in a lack of sustainability in the infection process; if the hydrogel network is too dense, bacteria may have difficulty contacting the bone marrow cavity tissue in a timely manner, potentially leading to insufficient infection initiation.

[0007] For osteomyelitis modeling in healthy mice, an ideal material system should not only allow some bacteria to be exposed early to initiate infection, but also retain another portion of bacteria as a reservoir, gradually releasing them after the formation of the infection microenvironment to simulate the pathological process of continuous colonization and progressive aggravation of osteomyelitis. Currently, there is a lack of a dual-reservoir type bacterial hydrogel system that combines initial infection initiation and subsequent reservoir release. Simultaneously, the intramedullary modeling material also needs to possess good bone interface retention capabilities. Ordinary hydrogels are easily displaced in the medullary cavity or bone defect area due to local fluid erosion, tissue movement, or pressure changes, resulting in bacteria or loads not being stably retained at the target site. If the material can form adhesion, coordination, or multi-point interactions with bone mineral surfaces, it helps to enhance its fixation effect in the medullary cavity and improve the consistency of local infection induction. DOPA groups containing catechol structures can generate strong interactions with inorganic mineral surfaces, metal ions, and hydroxyl-containing surfaces, and therefore can be used to improve the adhesion properties of materials to bone tissue or bone mineral interfaces. However, among existing osteomyelitis modeling materials, there are still few technical solutions that integrate a temperature-sensitive bone adhesion matrix, a pathogen enzyme-responsive crosslinking network, and an acid-sensitive bone mineralization microcavity for osteomyelitis modeling in healthy mice.

[0008] In summary, existing mouse osteomyelitis modeling techniques have at least the following shortcomings: First, simple bacterial suspension injection is prone to bacterial leakage and uneven local distribution, resulting in insufficient model stability; Second, ordinary bacterial hydrogels are mostly released in a single instance or passively, making it difficult to simulate the gradual progression and continuous colonization of infection; Third, existing stimulus-responsive materials mostly rely on single pH, single enzyme, or single temperature responses, lacking a combined response mechanism targeting the acidified environment of osteomyelitis and Staphylococcus aureus pathogen signals; Fourth, existing materials lack a clear dual-repository design of initiating and reservoir bacteria, making it difficult to simultaneously address infection initiation and subsequent infection maintenance; Fifth, ordinary hydrogels have limited adhesion and local retention capabilities at the bone mineral interface, which is not conducive to forming a stable infection microenvironment in the confined space of the mouse bone marrow cavity.

[0009] Therefore, there is an urgent need to develop a novel injectable hydrogel system for modeling osteomyelitis in healthy mice. This system should maintain good fluidity at low temperatures for easy introduction into the medullary cavity; it should be able to gel in situ at body temperature to achieve local bacterial confinement; it should have bone adhesion capabilities to enhance material retention in the medullary cavity and on bone mineral surfaces; it should be able to separate Staphylococcus aureus into two parts: a continuous initiating bacterium and a microcavity reservoir bacterium, enabling the initiating bacterium to induce local infection in the early stages, and the reservoir bacterium to be released in stages after the formation of the infection microenvironment; simultaneously, the system should also be able to respond to osteomyelitis-related acidification environments and Staphylococcus aureus-related pathogenic enzyme signals to achieve a self-triggered release that more closely matches the pathological process of osteomyelitis. Summary of the Invention

[0010] To address the technical problems of existing mouse osteomyelitis modeling methods, such as easy leakage of bacterial solution, lack of persistence of infection and pathological correlation, and lack of bacterial hydrogel systems with bone adhesion, temperature-sensitive gelation, dual acid and enzyme response and dual library release functions, this invention provides an injectable gel for osteomyelitis model construction and its preparation method.

[0011] According to one aspect of the present invention, a method for preparing an injectable gel for constructing an osteomyelitis model is provided, comprising: S1, Preparation of bone adhesion thermosensitive polyphosphononitrile PPZ-DOPA: Hexachlorocyclotriphosphononitrile was obtained by thermal ring-opening polymerization to polydichlorophosphononitrile; L-isoleucine was esterified with thionyl chloride and ethanol to obtain L-isoleucine ethyl hydrochloride; L-DOPA was esterified with thionyl chloride and methanol to obtain L-DOPA methyl hydrochloride; monomethoxy polyethylene glycol was reacted with sodium hydride to obtain mPEG sodium salt; polydichlorophosphononitrile was dissolved in anhydrous tetrahydrofuran and subjected to side chain substitution reactions with mPEG sodium salt, L-isoleucine ethyl hydrochloride and L-DOPA methyl hydrochloride in sequence, followed by precipitation, purification, dialyzing and drying to obtain PPZ-DOPA; S2, to prepare pullulan oxide OPul; Pullulan was dissolved in water, sodium periodate was added in the dark, and ethylene glycol was added to terminate the reaction after the reaction was completed. The product was then dried by dialysis to obtain OPul. S3, preparation of Pep-DH, a cross-linking agent for pathogen-enzyme-cleavable diacylhydrazide peptides; After swelling of 2-chlorotriphenylmethyl chloro resin in anhydrous DMF, Fmoc-Gly-OH, DIPEA, and DMAP were added. Following the reaction and washing, unreacted active sites of the resin were blocked with a methanol / DIPEA / DMF blocking solution. Using a solid-phase synthesis method with Fmoc, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Gly-OH were sequentially coupled to the resin via a deprotection-coupling cycle to obtain a peptide intermediate containing the Gly-Glu-Glu-Gly sequence. The terminal Fmoc protecting group was removed with piperidine / DMF. Using DMF as a solvent, succinic anhydride and DIPEA were added to introduce a succinyl group at the N-terminus of the peptide chain. The peptide chain was cleaved using a lysis buffer, and the side chain protecting groups were removed to obtain a dicarboxylated peptide intermediate. The dicarboxylated peptide intermediate was dissolved in a dimethyl sulfoxide / water mixture, activated with EDC·HCl and NHS, and then ADH was added. After dialyzing and drying, Pep-DH was obtained. S4, preparation of Staphylococcus aureus reservoir-type bone mineralization microcavities Sau-Sr / Mg-CaP-MC; Sodium alginate and gelatin were dissolved in water and mixed with a Staphylococcus aureus suspension to obtain a precursor containing a bacterial core. This precursor was then dropped into a calcium ion crosslinking solution and solidified to form bacterial calcium alginate-gelatin soft microspheres. After washing, bacterial Alg-Gel soft cores were obtained. The bacterial Alg-Gel soft cores were then incubated sequentially in OPul solution and Pep-DH solution to form an acylhydrazone crosslinking membrane with Pep-DH, resulting in a microcavity intermediate with an outer layer that could be cleaved by OPul / Pep-DH pathogenic enzymes. The microcavity intermediate was then alternately placed in mineralization solution A containing calcium, strontium, and magnesium ions and mineralization solution B containing phosphate ions for in-situ deposition of an Sr / Mg-doped amorphous calcium phosphate layer on the surface. After several cycles, Sau-Sr / Mg-CaP-MC microcavities were obtained. S5, Preparation of Sau-DualGel, an injectable hydrogel loaded with Staphylococcus aureus dual library; Under conditions of 2–8℃, PPZ-DOPA and OPul are dissolved in pre-cooled sterile buffer to obtain phase A, and Pep-DH and Sau-Sr / Mg-CaP-MC microcavities are dissolved in pre-cooled sterile buffer to obtain phase B. Staphylococcus aureus bacterial suspension is added to phase A as the continuous phase starter bacteria and then mixed with phase B to obtain Sau-DualGel precursor. The Sau-DualGel precursor is a flowable sol at 2–8℃ and gels in situ at 30–37℃.

[0012] Preferably, the hexachlorocyclotriphosphononitrile in S1 is purified before thermal ring-opening polymerization: the hexachlorocyclotriphosphononitrile is dissolved by heating and reflux in anhydrous n-hexane, and the insoluble impurities are removed by hot filtration. The filtrate is cooled and allowed to stand to crystallize. The crystals are collected by filtration and recrystallized. After drying, purified hexachlorocyclotriphosphononitrile is obtained.

[0013] Preferably, the specific steps of the side chain substitution reaction in S1 include: cooling the polydichlorophosphononil tetrahydrofuran solution to 0-5°C under nitrogen protection, adding mPEG sodium salt solution dropwise, raising the temperature to room temperature after the reaction and continuing the reaction, then adding L-isoleucine ethyl ester hydrochloride and triethylamine, stirring the reaction at room temperature, then adding L-DOPA methyl ester hydrochloride and triethylamine, raising the temperature to continue the reaction, cooling to room temperature after the reaction is completed, filtering to remove solid byproducts, concentrating the filtrate under reduced pressure and then dropping it into pre-cooled anhydrous diethyl ether to precipitate, and purifying, dialyzing and drying the precipitate to obtain bone adhesion thermosensitive polyphosphononil PPZ-DOPA.

[0014] Preferably, in the PPZ-DOPA, the molar ratio of monomethoxy polyethylene glycol, L-isoleucine ethyl ester hydrochloride and L-DOPA methyl ester hydrochloride is (30-60):(35-65):(1-10).

[0015] Preferably, in the solid-phase synthesis of Fmoc in S3, deprotection is carried out by a piperidine / DMF solution oscillation reaction to remove the Fmoc protecting group; coupling is carried out by adding Fmoc protected amino acid, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and DIPEA as reaction solvent and oscillating reaction to couple new amino acid residues; washing with DMF after each deprotection and coupling.

[0016] Preferably, in S3, the volume ratio of methanol, DIPEA, and DMF in the methanol / DIPEA / DMF blocking solution is 5:5:90; the pyrolysis solution is a mixed solution of TFA / TIS / water with a volume ratio of 95:2.5:2.5.

[0017] Preferably, in S4, the calcium ion crosslinking solution is a CaCl2 solution; the mineralization solution A is a HEPES buffer containing CaCl2, SrCl2, and MgCl2; and the mineralization solution B is a HEPES buffer containing Na2HPO4.

[0018] Preferably, in S5, the pH of phase A is controlled at 5.8 to 6.8, and after phase A and phase B are mixed, the overall pH is controlled at 6.5 to 7.2.

[0019] Preferably, the content of Sau-Sr / Mg-CaP-MC microcavities in the Sau-DualGel precursor described in S5 is 5–15 mg / mL.

[0020] According to another aspect of the present invention, an injectable gel for constructing an osteomyelitis model is provided, which is prepared by the method for preparing an injectable gel for constructing an osteomyelitis model as described above.

[0021] The beneficial effects of this invention are: (1) This invention designs Staphylococcus aureus as a dual-repository loading form of continuous phase initiating bacteria and microcavity reservoir bacteria, so that the initiating bacteria can first induce local initial infection in the bone marrow cavity of healthy mice, and then the reservoir bacteria are released in stages after the formation of the infection microenvironment. This improves the problems of easy escape, easy dilution, uneven local distribution and large fluctuations in the infection process after a single injection of traditional bacterial suspensions, and is conducive to improving the stability, persistence and repeatability of mouse osteomyelitis modeling.

[0022] (2) This invention constructs a bone acid-pathogen enzyme dual-key-triggered release mechanism. The calcium-phosphorus mineralized shell of the reservoir bacteria microcavity can gradually dissolve in the osteomyelitis-associated acidification environment, while the inner pathogen enzyme-cleaved cross-linked membrane can be loosened under the action of Staphylococcus aureus-associated protease. The two work synergistically to promote the responsive release of reservoir bacteria in the infection microenvironment. Compared with single pH response, single enzyme response, or ordinary passive diffusion hydrogels, the release process of this invention can better simulate the pathological evolution of osteomyelitis from the initiation of local infection, the formation of the infection microenvironment, to the continuous development of infection.

[0023] (3) This invention uses bone adhesion thermosensitive polyphosphononitrile as the main matrix of the hydrogel, which keeps the system in an injectable sol state at low temperatures and gels in situ at body temperature. The hydrogel's local retention capacity within the bone marrow cavity is improved through coordination, hydrogen bonding, and multi-point adsorption between the catechol groups in the DOPA side chains and calcium ions, phosphate groups, and hydroxyl structures on the bone mineral surface. This structure helps reduce the escape of bacterial-carrying materials from the target bone cavity, enhances the local infection confinement effect, and further improves the consistency and reliability of the osteomyelitis model construction. Attached Figure Description

[0024] 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 This is an electron microscope image of pullulan oxide OPul from the present invention; Figure 2 This is a Pep-DH electron microscope image of the present invention; Figure 3 Electron micrograph of the microcavity intermediate of the present invention having an OPul / Pep-DH pathogenic enzyme capable of cleaving the inner membrane; Figure 4 This is an electron microscope image of a reservoir-type microcavity with a Sr / Mg-doped amorphous calcium phosphate layer according to the present invention. Figure 5 This is an electron microscope image of the Sau-DualGel precursor of this invention; Figure 6 This is a diagram showing the in-situ gelation effect of the Sau-DualGel precursor of this invention at 37°C. Figure 7 This is an image showing the injection effect of the Sau-DualGel precursor of the present invention; Figure 8 This is an image of the Sau-DualGel precursor injected into the bone marrow cavity of mice according to the present invention; Figure 9 This is a local infection observation diagram of a mouse osteomyelitis model 21 days after injection of the Sau-DualGel precursor of this invention. Figure 10 This is a graph showing the changes in local bacterial load in different groups of osteomyelitis models according to the present invention; Figure 11 This image shows the observation of local infection in a mouse osteomyelitis model after 21 days of different molar ratios of monomethoxy polyethylene glycol mPEG550, L-isoleucine ethyl ester hydrochloride, and L-DOPA methyl ester hydrochloride. In the image, the molar ratio of the feed is 30:35:1, the molar ratio of the feed is 45:50:5, and the molar ratio of the feed is 60:65:10. Figure 12 This is a graph showing the changes in local bacterial load in osteomyelitis models with different microcavity contents according to the present invention. Detailed Implementation

[0025] 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.

[0026] Unless otherwise specified, all conditions in the examples were performed under standard conditions. Reagents or instruments whose manufacturers are not specified are commercially available products. Unless otherwise stated, all technical and scientific terms herein have the meanings commonly understood by one of ordinary skill in the art.

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0028] Example 1: Preparation of bone-adhesive thermosensitive polyphosphononitrile PPZ-DOPA.

[0029] This embodiment prepares a polyorganophosphononil material, denoted as PPZ-DOPA, which simultaneously possesses low-temperature solubility, body-temperature gelation, biodegradability, and bone mineral adhesion capabilities. This material serves as the main temperature-sensitive matrix of the composite hydrogel, ensuring that the hydrogel precursor is injectable at low temperatures, rapidly gels in situ after entering the body, and enhances its retention capacity in the medullary cavity and bone mineral interface through DOPA side chains.

[0030] Step 1.1: Take 10.0 g of hexachlorocyclotriphosphononitrile and add it to a 250 mL three-necked flask, then add 150 mL of anhydrous n-hexane. Connect the flask to a condenser and heat to 68–70 °C under nitrogen protection, refluxing at 400 rpm for 1 h to ensure complete dissolution of the hexachlorocyclotriphosphononitrile. While hot, filter through a preheated sintered glass funnel to remove insoluble impurities. After the filtrate cools naturally to room temperature, let it stand at 4 °C for 4 h to allow crystals to fully separate. Collect the crystals by filtration and recrystallize them again with anhydrous n-hexane using the same method. Dry the resulting crystals in a vacuum oven at 40 °C and a vacuum degree not lower than -0.08 MPa for 12 h to obtain purified hexachlorocyclotriphosphononitrile.

[0031] Step 1.2: Take 6.0 g of purified hexachlorocyclotriphosphononitrile and add it to a flame-dried glass polymerization tube. After vacuuming and purging with nitrogen three times, the polymerization tube is sealed under vacuum. The sealed polymerization tube is placed in an oil bath at 250℃ and heated for 8 hours to allow the hexachlorocyclotriphosphononitrile to undergo thermal ring-opening polymerization, generating polydichlorophosphononitrile. This reaction is a melt ring-opening polymerization, and no mechanical stirring is required during the reaction. After the reaction, the polymerization tube is allowed to cool naturally to room temperature. The polymerization tube is then opened, and 120 mL of anhydrous tetrahydrofuran is added. The mixture is stirred at 300 rpm for 4 hours under nitrogen protection to ensure that the generated polydichlorophosphononitrile is fully dissolved. The resulting solution is filtered under nitrogen protection to remove insoluble matter, yielding a polydichlorophosphononitrile tetrahydrofuran solution. Because the P-Cl bond in polydichlorophosphononitrile is sensitive to moisture, it should not be exposed to air for extended periods and is directly used for the side-chain substitution reaction in step 1.6.

[0032] Step 1.3: Take 5.0 g of L-isoleucine and add it to a 250 mL three-necked flask, then add 100 mL of anhydrous ethanol. Stir at 300 rpm in an ice-water bath to form a suspension. Slowly add 8 mL of thionyl chloride dropwise over 30–45 min, keeping the reaction temperature below 10 °C during the addition. After the addition is complete, remove the ice-water bath, raise the temperature to room temperature and continue stirring for 2 h, then raise the temperature to 70 °C and reflux for 6 h, stirring at 400 rpm to ensure complete esterification of the L-isoleucine carboxyl group. After the reaction is complete, remove ethanol and excess thionyl chloride under reduced pressure below 40 °C. Add 100 mL of anhydrous diethyl ether to the residue, wash thoroughly by shaking, and filter. Repeat the ether washing three times. Place the obtained solid in a vacuum drying oven at 35 °C and dry for 12 h to obtain L-isoleucine ethyl ester hydrochloride.

[0033] Step 1.4: Take 3.0 g of L-DOPA and add it to a 250 mL three-necked flask. Add 80 mL of anhydrous methanol and stir at 300 rpm under nitrogen protection and an ice-water bath. Slowly add 5 mL of thionyl chloride to the system over 30 min, keeping the system temperature below 10 °C during the addition. After the addition is complete, raise the temperature to room temperature and stir for 2 h, then raise the temperature to 65 °C and reflux for 6 h, stirring at 350–450 rpm. After the reaction is complete, remove the solvent under reduced pressure below 40 °C. Add 100 mL of anhydrous diethyl ether to the residue to precipitate the solid. Filter and wash three times with anhydrous diethyl ether, then dry under vacuum at 35 °C for 12 h to obtain L-DOPA methyl ester hydrochloride.

[0034] Step 1.5: Take 8.0 g of monomethoxy polyethylene glycol (mPEG550) and add it to a dried three-necked flask. Add 80 mL of anhydrous tetrahydrofuran and stir to dissolve under nitrogen protection. Cool the system to 0–5 °C and add 0.45 g of sodium hydride in batches, maintaining a stirring speed of 300 rpm and controlling the system temperature to not exceed 10 °C. After the addition is complete, continue the reaction at 0–5 °C for 1 h, then raise the temperature to room temperature and react for 2 h to convert the terminal hydroxyl groups of mPEG to mPEG sodium salt, obtaining an mPEG sodium salt solution. The obtained solution is directly used for side chain substitution in step 1.6.

[0035] Step 1.6: Place the polydichlorophosphononil tetrahydrofuran solution obtained in Step 1.2 in a dry three-necked flask and cool it to 0–5°C under nitrogen protection, with the stirring speed controlled at 300 rpm. Slowly add the mPEG sodium salt solution obtained in Step 1.5 dropwise to the polydichlorophosphononil tetrahydrofuran solution through a constant pressure dropping funnel, with the addition time controlled at 1 h. After the addition is complete, continue the reaction at 0–5°C for 2 h, then raise the temperature to room temperature and react for 6 h, allowing some P-Cl bonds to be replaced by mPEG side chains. Then add 4.5 g of L-isoleucine ethyl ester hydrochloride and 6.0 mL of triethylamine obtained in Step 1.3, and stir at room temperature for 12 h at a stirring speed of 350 rpm. After that, add 0.65 g of L-DOPA methyl ester hydrochloride and 1.0 mL of triethylamine obtained in Step 1.4, raise the temperature of the system to 40°C, and continue the reaction for 36 h at a stirring speed controlled at 350–450 rpm. During the reaction, nitrogen or argon atmosphere was maintained, and the operation was carried out in the dark to reduce the risk of oxidation of the catechol structure in DOPA and hydrolysis of the P-Cl bonds in polydichlorophosphononitrile. After the reaction was completed, the reaction solution was cooled to room temperature and filtered to remove solid byproducts such as triethylamine hydrochloride. The filtrate was concentrated under reduced pressure at below 35°C to approximately 30–50 mL, and then slowly added dropwise to 800 mL of pre-cooled anhydrous diethyl ether. During the addition, the diethyl ether system was stirred at 500 rpm to ensure complete precipitation of the polymer. The precipitate was collected, redissolved in tetrahydrofuran, and then precipitated again with cold diethyl ether. This purification process was repeated three times. The resulting precipitate was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days at a temperature of 4–10°C, with the dialysate changed every 6–8 hours. After dialysis, the dialysate was freeze-dried for 48 hours to obtain bone-adhesive thermosensitive polyphosphononitrile PPZ-DOPA.

[0036] The working mechanism of PPZ-DOPA lies in the synergistic regulation of hydrogel behavior by its three types of side chains: the mPEG side chain enhances the polymer's solubility in low-temperature aqueous phases, enabling it to form a flowable sol at 2–8°C; the L-isoleucine ethyl ester side chain provides hydrophobic association upon temperature elevation, with hydrophobic segments aggregating to form physical cross-linking points when the system enters a body-temperature environment, driving the sol to transform into a gel; the DOPA methyl ester side chain contains catechol groups, which can form coordination, hydrogen bonding, and multi-point adsorption interactions with calcium ions, phosphate groups, and hydroxyl structures on the bone mineral surface, thereby enhancing the adhesion and retention of the hydrogel on the bone marrow cavity wall and bone mineral interface. Therefore, this polymer not only provides in-situ gelation capability but also plays a role in locally confining Staphylococcus aureus and reducing material leakage.

[0037] Example 2: Preparation of pullulan oxide OPul.

[0038] This embodiment prepares OPul, an aldehyde-containing polysaccharide for dynamic acylhydrazone crosslinking. OPul, as the aldehyde donor in the composite hydrogel of this invention, can undergo a reversible condensation reaction with Pep-DH, a pathogen enzyme-cleavable diacylhydrazine peptide crosslinking agent, to form a dynamic crosslinking network with pH sensitivity and pathogen enzyme response.

[0039] Step 2.1: Take 2.0g of pullulan polysaccharide and add it to a 500mL brown round-bottom flask. Then add 200mL of deionized water and stir magnetically at 400rpm for 4h at room temperature to completely dissolve the pullulan and obtain a pullulan solution with a mass concentration of 1wt%.

[0040] Step 2.2: Weigh 0.65g of sodium periodate and dissolve it in 20mL of deionized water in the dark. Slowly add the sodium periodate solution to the pullulan solution through a dropping funnel, controlling the addition time to be 10-15 minutes. After the addition is complete, wrap the reaction flask with aluminum foil to protect it from light, and continue stirring the reaction at 20-25℃ for 6 hours, controlling the stirring speed to be 350 rpm. During this process, sodium periodate selectively oxidizes the vicinal diol structure on the pullulan molecular chain, causing some sugar rings to open and generate aldehyde groups. The reaction time and the amount of sodium periodate used determine the degree of aldehyde formation. Too low an aldehyde formation rate will result in slow subsequent gelation and insufficient crosslinking density; too high an aldehyde formation rate will cause excessive degradation of the polysaccharide backbone, leading to a decrease in gel mechanical properties. Therefore, in this embodiment, the reaction time is controlled to be 6 hours, and the amount of sodium periodate is controlled to be 0.65g.

[0041] After the reactions in steps 2.3 and 2.2 are completed, 2 mL of ethylene glycol is added to the system, and the mixture is stirred at room temperature for 30 min at 300 rpm to consume the remaining sodium periodate and terminate the oxidation reaction. The reaction solution is then transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed with deionized water for 3 days at a temperature controlled between 4 and 10 °C. The dialysate is changed every 4 hours on the first day and every 8 hours thereafter. After dialysis, the dialysate is pre-frozen at -80 °C for 6 hours and then freeze-dried for 48 hours to obtain pullulan oxide OPul. Figure 1 As shown.

[0042] In the composite hydrogel, the aldehyde group of OPul forms an acylhydrazone bond with the hydrazide group of Pep-DH. Acylhydrazone bonds are dynamic covalent bonds, maintaining a certain degree of stability under weakly acidic to neutral conditions. As the ambient pH decreases, the dynamic exchange and dissociation tendency of acylhydrazone bonds increases, leading to the gradual loosening of the cross-linked network. Therefore, OPul not only participates in the formation of the hydrogel network but also endows the system with sensitivity to the acidified microenvironment of infection. When the local pH of osteomyelitis decreases, the acylhydrazone network gradually loosens, increasing the gel pore size, which is conducive to the release of reservoir bacteria and the continuous evolution of the infection process.

[0043] Example 3: Preparation of Pep-DH, a cross-linking agent for diacylhydrazide peptides that can be cleaved by pathogenic enzymes.

[0044] This embodiment prepares a cross-linking agent containing hydrazide groups at both ends and a pathogen enzyme-recognizable peptide segment in the middle, denoted as pathogen enzyme-cleavable dihydrazide peptide cross-linking agent (Pep-DH). This cross-linking agent is both a dynamic cross-linking agent for pullulan oxidase (OPul) and a response unit for the infection microenvironment. Its preferred structure is ADH-Suc-Gly-Glu-Glu-Gly-ADH, including a Gly-Glu-Glu-Gly peptide segment in the middle, a succinyl linker arm connected to one end of the peptide segment, and hydrazide terminal groups at both ends of the molecule. Here, ADH represents the hydrazide functional unit introduced by adipic acid dihydrazide, Suc represents the succinyl linker arm, and the Gly-Glu-Glu-Gly peptide segment contains glutamate residues, which can serve as potential cleavage sites for Staphylococcus aureus secreted proteases.

[0045] Step 3.1: Take 2.0 g of 2-chlorotriphenylmethyl chloride resin and add it to a solid-phase synthesis reaction column. Add 30 mL of anhydrous N,N-dimethylformamide (DMF) and swell the resin at room temperature using a solid-phase synthesis shaker for 30 min, with the shaking frequency controlled at 80–120 times / min. Add 1.2 g of N-[(9H-fluorene-9-ylmethoxy)carbonyl]glycine (Fmoc-Gly-OH), 2.0 mL of N,N-diisopropylethylamine (DIPEA), and 20 mg of 4-dimethylaminopyridine (DMAP), and react at room temperature for 4 h. After the reaction is complete, wash the resin 5 times with 20 mL of DMF each time. Then add 30 mL of blocking solution with a methanol / DIPEA / DMF volume ratio of 5:5:90 and react at room temperature for 30 min to block the unreacted resin active sites. After blocking, wash the resin 5 times with DMF.

[0046] Step 3.2: Peptide chain elongation was performed using the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis method. This method progressively links amino acids to the ends of resin-loaded peptide chains through a deprotection-coupling cycle. Before each round of coupling, 30 mL of a 20% piperidine / N,N-dimethylformamide (DMF) solution was added to the solid-phase synthesis column, and the reaction was carried out with shaking at room temperature for 20 min to remove the Fmoc protecting groups attached to the ends of the peptide chains, exposing the terminal amino groups. After deprotection, the deprotection solution was drained, and the resin was washed five times with DMF, adding 20–30 mL of DMF each time, shaking for 1–2 min, and then draining to remove residual piperidine and deprotection byproducts. The amino acid coupling reaction was then carried out. Based on the molar amount of reactive amino groups on the resin, approximately three times the molar amount of Fmoc-protected amino acids, approximately three times the molar amount of O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), approximately three times the molar amount of 1-hydroxybenzotriazole (HOBt), and approximately six times the molar amount of N,N-diisopropylethylamine (DIPEA) were added, along with DMF as the reaction solvent, to completely submerge the resin in the reaction solution. The aforementioned Fmoc-protected amino acids were activated under the action of the HBTU / HOBt / DIPEA system and underwent an amidation reaction with the terminal amino groups on the resin that had been deprotected, thereby attaching new amino acid residues to the peptide chain terminus. The coupling reaction was carried out at room temperature for 1.5–2 h, during which a solid-phase synthesis oscillator was used to oscillate at a frequency of 100 times / min to ensure sufficient contact between the resin particles and the reaction solution. Following the aforementioned deprotection and coupling cycle, N-[(9H-fluorene-9-ylmethoxy)carbonyl]-L-glutamic acid-5-tert-butyl ester (Fmoc-Glu(OtBu)-OH), Fmoc-Glu(OtBu)-OH, and N-[(9H-fluorene-9-ylmethoxy)carbonyl]glycine (Fmoc-Gly-OH) were sequentially coupled. In Fmoc-Glu(OtBu)-OH, OtBu is a tert-butyl ester protecting group for the glutamic acid side chain carboxyl group, used to prevent the glutamic acid side chain carboxyl group from participating in side reactions during peptide chain elongation. After each amino acid coupling, the resin was thoroughly washed five times with DMF to remove unreacted amino acids, condensing agents, bases, and byproducts. Through these steps, peptide intermediates containing the Gly-Glu-Glu-Gly (glycine-glutamic acid-glutamic acid-glycine) sequence were sequentially constructed on the resin. The glutamate residues in this peptide can provide potential recognition sites for proteases secreted by Staphylococcus aureus that have a preference for glutamate residue cleavage, making the resulting Pep-DH crosslinker more prone to peptide fragmentation in the presence of infection-related proteases.

[0047] After peptide chain elongation in steps 3.3 and 3.2, the terminal Fmoc protecting group was removed again using 20% ​​piperidine / DMF. Then, 0.8 g of succinic anhydride and 1.5 mL of DIPEA were added, and the mixture was reacted at room temperature for 3 h using 30 mL of DMF as solvent, to introduce a succinyl group at the N-terminus of the peptide chain. After the reaction was complete, the resin was washed sequentially with DMF, dichloromethane, and methanol, 3–5 times each, and then the resin was dried by blowing or vacuum drying for 30 min.

[0048] Step 3.4: Add 30 mL of lysis buffer (TFA / TIS / water volume ratio 95:2.5:2.5) to the dried resin. Incubate at room temperature with shaking for 2 h to lyse the peptide chains from the resin and remove side-chain protecting groups. After lysis, filter to remove the resin. Concentrate the filtrate under reduced pressure below 30°C to a small volume, then add dropwise to 300 mL of pre-cooled anhydrous diethyl ether. Let stand for 30 min to allow complete precipitation of the crude peptide. Collect the precipitate by centrifugation or filtration, wash three times with cold diethyl ether, and dry under vacuum to obtain the dicarboxylated peptide intermediate HOOC-Suc-Gly-Glu-Glu-Gly-COOH, where HOOC represents the carboxyl group.

[0049] Step 3.5: Take 500 mg of the dicarboxylic acid peptide intermediate obtained in Step 3.4 and add it to a 50 mL round-bottom flask. Add 20 mL of a 1:1 mixture of dimethyl sulfoxide (DMSO) and water, and stir at 300 rpm at room temperature until dissolved. Add 600 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 350 mg of N-hydroxysuccinimide (NHS), and activate at room temperature for 2 h with stirring at 300 rpm. Then add 2.0 g of adipic acid dihydrazide (ADH), and adjust the pH of the system to 5.8–6.2 with dilute hydrochloric acid or sodium hydroxide solution. React at room temperature for 18 h with stirring at 250–350 rpm. During the reaction, the carboxyl groups at both ends of the dicarboxylic acid peptide intermediate are activated by EDC·HCl / NHS and then undergo an amidation reaction with the hydrazide group at one end of excess ADH. The hydrazide group at the other end of the ADH remains as the active end group, which can subsequently undergo a condensation reaction with the aldehyde group on the OPul molecular chain to form an acylhydrazone bond. After the reaction, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 500-1000 Da and dialyzed with deionized water for 48 h. The dialysis temperature is controlled at 4-10 °C, and the dialysis water is changed every 4-6 h. After dialysis, the dialysate is freeze-dried for 48 h to obtain Pep-DH, as shown below. Figure 2 As shown.

[0050] The working mechanism of Pep-DH lies in the fact that the hydrazide groups at both ends of its molecule can form dynamic hydrazone bonds with the aldehyde groups on the OPul molecular chain, thereby connecting the OPul molecular chains into a three-dimensional cross-linked network. Its central Gly-Glu-Glu-Gly peptide segment contains an acidic amino acid sequence, which can serve as a recognition and cleavage site for Staphylococcus aureus-associated proteases. The Staphylococcus aureus-associated proteases preferably include gamma-glutamyl endopeptidase Glu-C secreted by Staphylococcus aureus. When local infection occurs, the levels of Staphylococcus aureus-secreted proteases and infection-associated proteases increase, and the Pep-DH intermediate peptide segment can undergo enzymatic cleavage, gradually reducing the cross-linking bridges that originally connected the OPul molecular chains, thereby decreasing the cross-linking density of the hydrogel, increasing network porosity, and promoting gel structure decomposition. This mechanism allows the hydrogel to degrade not simply by ordinary pH or temperature changes, but to respond to Staphylococcus aureus infection-related signals, thus enhancing the pathogen-related specificity of the modeling system.

[0051] Example 4: Preparation of Sau-Sr / Mg-CaP-MC, a reservoir-type bone mineralization microcavity of Staphylococcus aureus.

[0052] This embodiment prepares a reservoir-type bone mineralization microcavity, Sau-Sr / Mg-CaP-MC. This microcavity consists of a bacterial Alg-Gel soft core, an OPul / Pep-DH pathogenic enzyme-cleavable inner membrane, and a Sr / Mg-doped amorphous calcium phosphate-sensitive outer shell. It is used to encapsulate Staphylococcus aureus and opens in stages when infection acidification and pathogenic enzymes coexist.

[0053] Step 4.1: Take 1.5g of sodium alginate and 1.0g of gelatin, add them to a 250mL sterile flask, add 100mL of sterile deionized water, and stir to dissolve in a 40℃ water bath for 2 hours at a stirring speed of 350rpm to obtain a homogeneous alginate-gelatin core precursor solution. After cooling the solution to a temperature that does not affect the activity of Staphylococcus aureus and the gelatin has not gelled significantly, add the Staphylococcus aureus bacterial suspension to the core precursor solution and mix gently to obtain a bacterial core precursor.

[0054] Step 4.2: Place the bacterial core precursor obtained in Step 4.1 into a sterile syringe or sterile microdroplet generator, and drop it into a pre-cooled calcium ion crosslinking solution (100 mmol / L CaCl2 solution) at 4–10°C. After the droplets enter the calcium ion crosslinking solution, the sodium alginate molecules on the surface and inside of the droplets undergo ionic crosslinking with calcium ions, forming a three-dimensional calcium alginate network, transforming the originally liquid bacterial droplets into bacterial calcium alginate-gelatin soft microspheres. To ensure uniform microsphere morphology, the collected solution is gently stirred at 100–200 rpm during the microsphere formation process to prevent droplets from sticking together or depositing. After all the microdroplets are added, continue to solidify in the calcium ion crosslinking solution for 10–20 minutes to allow calcium ions to further diffuse into the interior of the microspheres and complete the crosslinking. After solidification, the obtained soft microspheres were collected and gently washed three times with sterile buffer to remove free components on the surface of the microspheres, thus obtaining bacteria-containing Alg-Gel soft cores.

[0055] This step utilizes the rapid ionic cross-linking reaction between sodium alginate and calcium ions to encapsulate Staphylococcus aureus dispersed in the liquid precursor within microspheres. The resulting Alg-Gel soft core serves as the first layer of support structure for the reservoir bacteria. The calcium alginate network provides the basic morphology and initial stability of the microspheres, while the gelatin component enhances their flexibility and hydrophilicity, allowing the microspheres to maintain structural integrity during subsequent encapsulation with the OPul / Pep-DH inner membrane and the Sr / Mg-CaP mineralized outer shell. This soft core is not intended for the immediate release of all bacteria but rather serves as the basic encapsulation space for the reservoir bacteria, providing the core structure for subsequent infection microenvironment-triggered release.

[0056] Step 4.3: The bacterial Alg-Gel soft core obtained in Step 4.2 is dispersed in a 1.0 wt% OPul solution and gently incubated for 20 min at pH 6.0 and 4–10 °C, with stirring speed controlled at 80–120 rpm, allowing OPul to be adsorbed and partially penetrated into the surface of the soft core. Then, 0.5 wt% Pep-DH solution is added, and incubation continues for 30–120 min at pH 5.5–6.5, allowing the aldehyde groups of OPul and the acylhydrazide groups of Pep-DH to gradually condense, forming an acylhydrazone cross-linked membrane. This process is repeated once to adjust the inner membrane thickness. After each coating, the membrane is gently washed with sterile buffer to remove free OPul and Pep-DH. The resulting microcavity intermediate outer layer contains OPul / Pep-DH pathogenic enzymes that can cleave the inner membrane, such as… Figure 3 As shown. This inner membrane restricts the escape of reservoir bacteria in the absence of infection signals, but gradually loosens due to the cleavage of Pep-DH in the presence of pathogenic enzymes.

[0057] Step 4.4: Constructing the Sr / Mg-doped amorphous calcium phosphate shell. Mineralization solution A: Dissolve CaCl2 in sterile HEPES buffer to a final concentration of 20 mmol / L; add SrCl2 to a final concentration of 1 mmol / L; add MgCl2 to a final concentration of 1 mmol / L; adjust the pH to 7.4. Preferably, mineralization solution A does not use a phosphate-containing PBS buffer system to avoid premature precipitation of calcium, strontium, or magnesium ions with phosphate before contact with the microcavity. Mineralization solution B: Dissolve Na2HPO4 in sterile HEPES buffer to a final concentration of 20 mmol / L, and adjust the pH to 7.4. Place the OPul / Pep-DH-coated microcavity intermediate in mineralization solution A and incubate at 4–10°C for 10 min with gentle stirring at 80–120 rpm to allow calcium, strontium, and magnesium ions to adsorb onto the microcavity surface and the cross-linked membrane structure. The microcavities were then collected, gently washed once with buffer solution, and then transferred to mineralization solution B. They were incubated at 4–10°C for 10 min to allow phosphate ions and adsorbed divalent metal ions to deposit in situ on the microcavity surface, forming an Sr / Mg-doped amorphous calcium phosphate layer. Figure 4 As shown. The above alternating treatment process of mineralizing solution A and mineralizing solution B is repeated for 2 to 4 cycles to obtain Sau-Sr / Mg-CaP-MC microcavities.

[0058] The working mechanism of this microcavity is a synergistic release regulation of an acid-responsive outer shell and an enzyme-responsive inner membrane. The outer Sr / Mg-CaP shell mimics bone mineral composition and remains relatively stable under near-neutral conditions, preventing rapid release of reservoir bacteria in the initial stage. When local infection leads to acidification, the calcium-phosphorus shell dissolves, exposing the inner OPul / Pep-DH membrane. The inner OPul / Pep-DH membrane acts as a pathogen enzyme response barrier, still restricting the release of reservoir bacteria in the absence or inadequacy of pathogen enzymes. Once Staphylococcus aureus infection is established and related proteases are produced, the Pep-DH peptide is cleaved, the cross-linking density of the inner membrane decreases, and the reservoir bacteria are gradually released. Therefore, this microcavity is not a single pH-responsive carrier, but rather exhibits a dual-response release characteristic: the acidic bone infection microenvironment promotes the dissolution of the outer calcium-phosphorus shell, while the pathogen enzyme promotes the loosening of the inner OPul / Pep-DH membrane.

[0059] Example 5: Preparation of Sau-DualGel, an injectable hydrogel loaded with Staphylococcus aureus.

[0060] This embodiment prepares a Staphylococcus aureus dual-reservoir loaded injectable hydrogel precursor, denoted as Sau-DualGel precursor, for final use in mouse osteomyelitis modeling. The Staphylococcus aureus in this precursor consists of two parts: a continuous initiating bacterium and a microcavity reservoir bacterium. The initiating bacterium is used to establish the initial infection, while the reservoir bacterium is released in stages after the formation of the infection microenvironment, thereby creating a continuous, self-progressing osteomyelitis modeling process.

[0061] Step 5.1: Take 150 mg of PPZ-DOPA prepared in Example 1, add it to 1 mL of pre-cooled sterile HEPES buffer, and stir to dissolve at 2–8°C for 4–6 h at a stirring speed of 250–350 rpm to obtain a homogeneous thermosensitive polyphosphononium solution. Add 20 mg of OPul prepared in Example 2 to the solution, and continue stirring at 2–8°C for 1 h to uniformly disperse or dissolve OPul, obtaining phase A.

[0062] Step 5.2: Take 4 mg of Pep-DH prepared in Example 3 and add it to 1 mL of pre-cooled sterile HEPES buffer. Gently stir at 2–8°C for 30 min to ensure complete dissolution. Then add 20 mg of Sau-Sr / Mg-CaP-MC microcavities prepared in Example 4 and gently stir or slowly tumble at a low speed of 80–120 rpm for 5–10 min to obtain phase B. Low-speed mixing helps maintain the integrity of the microcavities and avoids microcavity rupture or premature escape of reservoir bacteria due to high shear.

[0063] The pH of phase A is controlled at 5.8–6.8 to reduce the possibility of premature cross-linking of OPul and Pep-DH before mixing and to maintain the fluidity of the precursor; the overall pH of phase A and phase B after mixing is controlled at 6.5–7.2 to balance the activity of Staphylococcus aureus, the stability of the calcium phosphate shell and the rate of acylhydrazone cross-linking reaction.

[0064] Step 5.3: A Staphylococcus aureus suspension was added to phase A as the continuous phase starter and gently mixed. Then, phase A containing the starter was mixed with phase B containing Sau-Sr / Mg-CaP-MC microcavities and Pep-DH at a volume ratio of 1:1 and gently mixed at 2–8°C to obtain the Sau-DualGel precursor. Its microstructure (SEM image) is shown below. Figure 5 The resulting precursor contained approximately 7.5 wt% PPZ-DOPA, 1.0 wt% OPul, and 0.2 wt% Pep-DH, with a Sau-Sr / Mg-CaP-MC microcavity content of 10 mg / mL. This precursor remained in a flowable sol state at 2–8 °C and underwent in-situ gelation at 30–37 °C. Figure 6 This demonstrates the in-situ gelation effect of the Sau-DualGel precursor at 37°C.

[0065] The working mechanism of Sau-DualGel consists of three consecutive stages. The first stage is the pre-injection stabilization stage. Under low temperature (2–8°C) and weakly acidic to near-neutral conditions, PPZ-DOPA is in a dissolved state, the reaction rate of OPul with Pep-DH acylhydrazone is low, and the system remains fluid, facilitating its introduction as a precursor (e.g., ...). Figure 7 The second stage is the thermogelation and bacterial confinement stage. After the precursor enters the body, the increased temperature causes the hydrophobic amino acid ester side chains of PPZ-DOPA to associate, forming a physical cross-linked network; simultaneously, the OPul aldehyde group and the Pep-DH acylhydrazine group form dynamic acylhydrazone bonds, constructing a chemical cross-linked network. After the two types of networks interpenetrate, both the continuous-phase initiating bacteria and the microcavity reservoir bacteria are confined to the local bone marrow cavity space. The third stage is the infection microenvironment response and release stage. The continuous-phase initiating bacteria first contact the local tissue and induce initial infection; as the infection progresses, local acidification and pathogen enzyme levels increase. Acidification dissolves the outer Sr / Mg-CaP shell of the microcavity, and the acidic environment enhances the dynamic dissociation tendency of the OPul / Pep-DH acylhydrazone bonds. The pathogen enzymes further cleave the Pep-DH peptide cross-linking bridges in the OPul / Pep-DH inner membrane and the main gel network, causing the microcavity inner membrane and gel network to gradually loosen, and the reservoir bacteria are released in stages. The released reservoir bacteria can further maintain the level of local infection and promote the continuous formation of the infection microenvironment, which is beneficial for constructing an osteomyelitis model with characteristics of continuous infection progression.

[0066] Through the above design, Sau-DualGel differs from ordinary bacterial suspension direct introduction methods and simple bacterial-loaded hydrogels. Ordinary bacterial suspensions lack local confinement and staged release mechanisms, making them susceptible to leakage, uneven distribution, and local residue differences; simple bacterial-loaded hydrogels typically only achieve initial embedding and are difficult to achieve secondary release of reservoir bacteria according to infection progression. In this embodiment, Sau-DualGel forms a dual-reservoir release structure by exposing the initiating bacteria in a continuous phase and delaying the release of reservoir bacteria through microcavities. Furthermore, it forms a dual-key-gated release logic through the bone acid-responsive outer shell and the pathogen enzyme-responsive inner membrane, thereby improving the persistence, locality, and consistency of disease progression in model establishment.

[0067] Example 6: Application of Sau-DualGel in mouse osteomyelitis modeling.

[0068] This embodiment is intended to illustrate the use of the Sau-DualGel precursor described in Example 5 in the induction of osteomyelitis in healthy mice.

[0069] During use, the Sau-DualGel precursor should be kept in a low-temperature, flowable state and introduced into the target medullary cavity or pre-defined bone defect area of ​​experimental mice under aseptic conditions. Figure 8The image shows the injection of Sau-DualGel precursor into the bone marrow cavity of mice. After entering the bone marrow cavity, PPZ-DOPA undergoes thermosensitive gelation under body temperature, forming the first layer of physical confinement network. Simultaneously, OPul and Pep-DH form dynamic acylhydrazone crosslinks, constructing the second layer of chemical network. Thus, both the initiating Staphylococcus aureus dispersed in the continuous phase and the reservoir Staphylococcus aureus embedded in the microcavity are locally confined within the bone marrow cavity.

[0070] In the initial stage of gel formation, the initiating bacteria in the continuous phase preferentially contact the local tissues of the bone marrow cavity and initiate local infection. As *Staphylococcus aureus* colonizes locally, its metabolic activity increases, and a host inflammatory response occurs, the local microenvironment gradually becomes acidic, accompanied by an increase in the activity of *Staphylococcus aureus*-secreted proteases and infection-related proteases. The acidic environment first induces the dissolution of the calcium phosphate shell of the Sau-Sr / Mg-CaP-MC microcavity. Subsequently, pathogenic enzymes cleave the Pep-DH peptide in the OPul / Pep-DH inner membrane, reducing the cross-linking density of the inner membrane and gradually releasing the reservoir of *Staphylococcus aureus*. Simultaneously, the OPul / Pep-DH cross-linking points in the hydrogel main network gradually loosen due to the acidic environment and the action of pathogenic enzymes, increasing gel pore size and promoting the diffusion of reservoir bacteria into the surrounding bone marrow tissue.

[0071] Therefore, this invention forms the following continuously progressive modeling mechanism: the material working mechanism of Sau-DualGel includes three consecutive stages. The first stage is the pre-injection stabilization stage. Under low temperature conditions of 2-8℃, PPZ-DOPA maintains a good aqueous dispersion or dissolution state, the acylhydrazone reaction rate between OPul and Pep-DH is relatively low, and the system as a whole maintains an injectable flowable state. The second stage is the thermogelation and local bacterial confinement stage. After the precursor enters the body, the temperature rise causes the hydrophobic amino acid ester side chains in PPZ-DOPA to undergo hydrophobic association, forming a physical cross-linking network; at the same time, the aldehyde group of OPul and the acylhydrazine group of Pep-DH gradually form dynamic acylhydrazone bonds, constructing a chemical cross-linking network. The physical cross-linking network and the dynamic covalent cross-linking network work together to confine the continuous phase initiating bacteria and the microcavity reservoir bacteria to the local space of the bone marrow cavity. The third stage is the infection microenvironment response and release stage. The continuous phase initiating bacteria preferentially contact the local tissue and induce the initial infection; as the infection progresses, the local microenvironment may show an acidification trend, accompanied by an increase in the levels of Staphylococcus aureus secreted proteases and infection-related proteases. The acidic microenvironment promotes the gradual dissolution of the outer Sr / Mg-CaP shell of the microcavity, while pathogenic enzymes facilitate the unwinding of Pep-DH peptide cross-linking bridges in the OPul / Pep-DH inner membrane and master gel network, leading to the phased release of reservoir bacteria. This process is beneficial for establishing an animal model of osteomyelitis that bridges the gap between early infection initiation and mid-to-late-stage infection maintenance.

[0072] Example 7: The effect of different bacterial loading methods on the persistence of osteomyelitis modeling.

[0073] To verify the advantages of the dual-library bacterial hydrogel of this invention in constructing an animal model of osteomyelitis, comparative experiments were conducted with a dual-library group, a single-library-full-initiation group, a single-library-full-reservoir group, and a free bacterial group. The total amount of Staphylococcus aureus used in each group was kept consistent, with only the bacterial loading method differing. The number of experimental animals, initial inoculation amount, sampling site, and CFU (colony-forming unit) counting method remained consistent across groups. Experimental results are expressed as mean ± standard deviation, and inter-group comparisons were performed using appropriate statistical methods.

[0074] Group A is a dual-reservoir group, which uses the Sau-DualGel described in this invention, namely a bone adhesion dual-key trigger type microcavity carrier hydrogel containing both continuous phase initiating bacteria and reservoir bacteria microcavities. Group B is the single-library-full-start group, in which the entire bacterial quantity is dispersed as the starter bacteria in the PPZ-DOPA / OPul / Pep-DH gel matrix, without setting up a microcavity for the storage bacteria; Group C is a single-library-full-library group, in which all bacterial counts are encapsulated in Sau-Sr / Mg-CaP-MC microcavities and then dispersed in PPZ-DOPA / OPul / Pep-DH gel matrix, without setting a continuous phase starter bacteria; Group D was the free bacteria group, and an equal volume of Staphylococcus aureus bacterial suspension was used directly for modeling.

[0075] Among them, the PPZ-DOPA / OPul / Pep-DH gel matrix refers to a continuous phase hydrogel network formed by PPZ-DOPA, OPul and Pep-DH.

[0076] Postoperatively, bone tissue or tissue samples from the infected area were collected on days 1, 3, 7, 14, and 21 for colony-forming unit (CFU) counting to evaluate the dynamic changes in local bacterial load under different bacterial loading methods. The results of local infection observation in the mouse osteomyelitis model on day 21 after injection of Sau-DualGel precursor are as follows: Figure 9 As shown. The relevant results are as follows. Figure 10 As shown, a high local bacterial load was detected in Group A on the first day after surgery, and then increased on the third and seventh days, reaching a high level on the seventh day; on the 14th and 21st days, the CFU in Group A remained at a relatively high level, indicating that the dual-liquidity bacterial-carrying hydrogel of the present invention is conducive to the formation of a continuous infection process that connects the early infection initiation and the maintenance of mid-to-late-stage infection.

[0077] Group B exhibited higher CFU levels in the early postoperative period, with significant bacterial load observed on days 1 and 3, indicating that using all bacteria as the continuous initiating bacteria could rapidly induce local infection. However, as time progressed, the CFU levels in Group B gradually decreased, significantly lower than those in Group A on days 14 and 21. This suggests that while relying solely on the initiating bacteria can lead to an early infection outbreak, the lack of subsequent reservoir bacteria makes it difficult to maintain long-term stable infection.

[0078] Group C showed lower CFU levels on postoperative days 1 and 3, indicating that when all bacteria were encapsulated within the microcavities, the early release of bacteria was limited due to the barrier effect of the outer mineralized shell and the inner responsive membrane, resulting in a slower infection initiation. As the modeling time increased, the CFU level in Group C gradually rose, reaching a relatively high level on day 14, indicating that the reservoir bacteria microcavities had a delayed release effect. However, due to the lack of a continuous phase initiating bacteria, early infection initiation in Group C was insufficient, and the overall modeling process lagged behind that of Group A.

[0079] In the early postoperative period, a certain bacterial load was detectable in group D (free bacterial suspension), even showing a moderately high level on day 1, but the margin of error was large, indicating significant inter-individual variability. As time progressed, the CFU level in group D decreased rapidly, showing significantly lower levels than in group A on days 7, 14, and 21. These results suggest that free bacterial suspensions lack gel confinement, bone adhesion retention, and reservoir release structures. After entering the bone marrow cavity, they may escape, spread, be diluted, or be cleared by local immune responses, making it difficult to maintain a stable local bacterial load and thus difficult to establish a stable, continuous, and reproducible osteomyelitis model.

[0080] In summary, the results show that Group A Sau-DualGel exhibits a typical two-stage infection dynamics of "early initiation and late maintenance"; Group B shows a single-exposure infection process of "high early stage and low late stage"; Group C shows a delayed release characteristic of "low early stage and delayed increase in late stage"; and Group D shows an unstable infection characteristic of "large fluctuations in early stage and rapid decline in late stage". Compared with single-storage bacterial gels and free bacterial suspensions, the dual-storage Sau-DualGel of this invention can rapidly establish local infection in the early stage through continuous phase initiating bacteria, and continuously release bacteria through the reservoir bacterial microcavities after the formation of the infection microenvironment, thereby maintaining a high and stable local bacterial load.

[0081] Therefore, the experimental results show that the Sau-DualGel described in this invention, through the synergistic effect between the continuous-phase initiating bacteria, the reservoir bacterial microcavity, the bone adhesion thermosensitive gel matrix, the pathogen enzyme-responsive inner membrane, and the bone acid-responsive mineralized outer shell, can improve the persistence, local stability, and reproducibility of infection during osteomyelitis modeling. It also improves upon the problems of large infection fluctuations, insufficient late-stage maintenance, or slow early initiation associated with traditional free bacterial injection and single-bacterial loading methods. These results further demonstrate that this invention is suitable for constructing stable, persistent, and reproducible osteomyelitis models in healthy mice.

[0082] Example 8: Experimental verification of different molar ratios of feed materials.

[0083] To verify the effects of monomethoxy polyethylene glycol mPEG550, L-isoleucine ethyl ester hydrochloride, and L-DOPA methyl ester hydrochloride at different molar ratios on the local infection status and bone defect repair in a mouse osteomyelitis model, this example involves injecting mice with different ratios of Sau-DualGel precursors into the bone marrow cavity and observing the local infection status 21 days later.

[0084] like Figure 11 As shown in Figure A, local infection observation in a mouse osteomyelitis model 21 days after injection of the Sau-DualGel precursor with a molar ratio of monomethoxy polyethylene glycol mPEG550, L-isoleucine ethyl ester hydrochloride, and L-DOPA methyl ester hydrochloride of 30:35:1; Figure B, local infection observation in a mouse osteomyelitis model 21 days after injection with a molar ratio of 45:50:5; and Figure C, local infection observation in a mouse osteomyelitis model 21 days after injection with a molar ratio of 60:65:10. The results showed that the bone defect areas in all three groups of mice had not healed after 21 days and remained infected. The results show that when the molar ratio of monomethoxy polyethylene glycol mPEG550, L-isoleucine ethyl ester hydrochloride and L-DOPA methyl ester hydrochloride is in the range of (30-60):(35-65):(1-10), Sau-DualGel can stably maintain the local infection microenvironment, which meets the requirements of this invention for the construction of an osteomyelitis model.

[0085] Example 9: Verification of the effects of different microcavity contents.

[0086] To determine the effect of Sau-Sr / Mg-CaP-MC microcavity content on the dynamic changes in local bacterial load and the duration of infection, this embodiment sets two microcavity contents (5 mg / mL and 15 mg / mL) of Sau-DualGel precursor, which are implanted locally in a mouse osteomyelitis model, and bacterial load changes are detected at different time points.

[0087] The results are as follows Figure 12As shown, when the Sau-Sr / Mg-CaP-MC microcavity content was 5 mg / mL, the reservoir bacterial count was low, allowing for early infection initiation. The bacterial load decreased on days 14 and 21, but remained at a high level. When the Sau-Sr / Mg-CaP-MC microcavity content was 15 mg / mL, the total reservoir bacterial count was high, with a high bacterial load in the mid-to-late stages that persisted for a longer period. Combined with... Figure 10 This indicates that when the Sau-Sr / Mg-CaP-MC microcavity content is in the range of 5–15 mg / mL, a high and stable local bacterial load can be maintained, which meets the requirements for constructing an osteomyelitis model.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. The present invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

[0089] The amounts of each material listed in the above embodiments can be scaled up or down proportionally in actual implementation, or adjusted within the conventional range; the given conventional operating conditions can also be appropriately adjusted according to the actual situation, or replaced with other conventional methods. For example, in the PPZ-DOPA obtained in Example 1, the mPEG side chain, hydrophobic amino acid ester side chain, and DOPA side chain together replace the P-Cl bond on the polyphosphazene main chain. In actual implementation, the proportion of the three types of side chains can be appropriately adjusted according to the target gelation temperature, adhesion performance, and water solubility; in step 3.1, in addition to oscillation swelling, mechanical stirring swelling is also performed, with the speed controlled at 150-250 rpm, which can also achieve the purpose of this invention; in step 4.2, the calcium ion crosslinking solution is a 5-200 mmol / L CaCl2 solution, which can also achieve the purpose of this invention.

Claims

1. A method for preparing an injectable gel for constructing an osteomyelitis model, characterized in that, include: S1, Preparation of bone adhesion thermosensitive polyphosphononitrile PPZ-DOPA: Hexachlorocyclotriphosphononitrile was obtained by thermal ring-opening polymerization to polydichlorophosphononitrile; L-isoleucine was esterified with thionyl chloride and ethanol to obtain L-isoleucine ethyl hydrochloride; L-DOPA was esterified with thionyl chloride and methanol to obtain L-DOPA methyl hydrochloride; monomethoxy polyethylene glycol was reacted with sodium hydride to obtain mPEG sodium salt; polydichlorophosphononitrile was dissolved in anhydrous tetrahydrofuran and subjected to side chain substitution reactions with mPEG sodium salt, L-isoleucine ethyl hydrochloride and L-DOPA methyl hydrochloride in sequence, followed by precipitation, purification, dialyzing and drying to obtain PPZ-DOPA; S2, to prepare pullulan oxide OPul; Pullulan was dissolved in water, sodium periodate was added in the dark, and ethylene glycol was added to terminate the reaction after the reaction was completed. The product was then dried by dialysis to obtain OPul. S3, preparation of Pep-DH, a cross-linking agent for pathogen-enzyme-cleavable diacylhydrazide peptides; After swelling of 2-chlorotriphenylmethyl chloro resin in anhydrous DMF, Fmoc-Gly-OH, DIPEA, and DMAP were added. Following the reaction and washing, unreacted active sites of the resin were blocked with a methanol / DIPEA / DMF blocking solution. Using a solid-phase synthesis method with Fmoc, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Gly-OH were sequentially coupled to the resin via a deprotection-coupling cycle to obtain a peptide intermediate containing the Gly-Glu-Glu-Gly sequence. The terminal Fmoc protecting group was removed with piperidine / DMF. Using DMF as a solvent, succinic anhydride and DIPEA were added to introduce a succinyl group at the N-terminus of the peptide chain. The peptide chain was cleaved using a lysis buffer, and the side chain protecting groups were removed to obtain a dicarboxylated peptide intermediate. The dicarboxylated peptide intermediate was dissolved in a dimethyl sulfoxide / water mixture, activated with EDC·HCl and NHS, and then ADH was added. After dialyzing and drying, Pep-DH was obtained. S4, preparation of Staphylococcus aureus reservoir-type bone mineralization microcavities Sau-Sr / Mg-CaP-MC; Sodium alginate and gelatin were dissolved in water and mixed with a Staphylococcus aureus suspension to obtain a precursor containing a bacterial core. This precursor was then dropped into a calcium ion crosslinking solution and solidified to form bacterial calcium alginate-gelatin soft microspheres. After washing, bacterial Alg-Gel soft cores were obtained. The bacterial Alg-Gel soft cores were then incubated sequentially in OPul solution and Pep-DH solution to form an acylhydrazone crosslinking membrane with Pep-DH, resulting in a microcavity intermediate with an outer layer that could be cleaved by OPul / Pep-DH pathogenic enzymes. The microcavity intermediate was then alternately placed in mineralization solution A containing calcium, strontium, and magnesium ions and mineralization solution B containing phosphate ions for in-situ deposition of an Sr / Mg-doped amorphous calcium phosphate layer on the surface. After several cycles, Sau-Sr / Mg-CaP-MC microcavities were obtained. S5, Preparation of Sau-DualGel, an injectable hydrogel loaded with Staphylococcus aureus dual library; Under conditions of 2–8℃, PPZ-DOPA and OPul are dissolved in pre-cooled sterile buffer to obtain phase A, and Pep-DH and Sau-Sr / Mg-CaP-MC microcavities are dissolved in pre-cooled sterile buffer to obtain phase B. Staphylococcus aureus bacterial suspension is added to phase A as the continuous phase starter bacteria and then mixed with phase B to obtain Sau-DualGel precursor. The Sau-DualGel precursor is a flowable sol at 2–8℃ and gels in situ at 30–37℃.

2. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, The hexachlorocyclotriphosphazene in S1 was purified before thermal ring-opening polymerization: Hexachlorocyclotriphosphononitrile was dissolved by heating and reflux in anhydrous n-hexane, and the insoluble impurities were removed by hot filtration. The filtrate was cooled and allowed to stand to crystallize. The crystals were collected by filtration and recrystallized, and then dried to obtain purified hexachlorocyclotriphosphononitrile.

3. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, The specific steps of the side chain substitution reaction in S1 include: The polydichlorophosphononitrile tetrahydrofuran solution was cooled to 0–5 °C under nitrogen protection, and mPEG sodium salt solution was added dropwise. After the reaction, the temperature was raised to room temperature and the reaction continued. Then, L-isoleucine ethyl ester hydrochloride and triethylamine were added, and the reaction was stirred at room temperature. Then, L-DOPA methyl ester hydrochloride and triethylamine were added, and the temperature was raised to continue the reaction. After the reaction was completed, the temperature was cooled to room temperature, and the solid byproducts were removed by filtration. The filtrate was concentrated under reduced pressure and then added dropwise to pre-cooled anhydrous diethyl ether to precipitate. The precipitate was purified, dialyzed, and dried to obtain bone adhesion thermosensitive polyphosphononitrile PPZ-DOPA.

4. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, In the PPZ-DOPA, the molar ratio of monomethoxy polyethylene glycol, L-isoleucine ethyl ester hydrochloride and L-DOPA methyl ester hydrochloride is (30-60):(35-65):(1-10).

5. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, In the solid-phase synthesis of Fmoc in S3, deprotection is carried out by oscillating a piperidine / DMF solution to remove the Fmoc protecting group; coupling is carried out by adding Fmoc protected amino acid, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and DIPEA as reaction solvent and oscillating to couple new amino acid residues; after each deprotection and coupling, the mixture is washed with DMF.

6. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, In S3, the volume ratio of methanol, DIPEA, and DMF in the methanol / DIPEA / DMF blocking solution is 5:5:90; the pyrolysis solution is a mixed solution of TFA / TIS / water with a volume ratio of 95:2.5:2.

5.

7. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, In S4, the calcium ion crosslinking solution is a CaCl2 solution; the mineralization solution A is a HEPES buffer containing CaCl2, SrCl2, and MgCl2; and the mineralization solution B is a HEPES buffer containing Na2HPO4.

8. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, In S5, the pH of phase A is controlled at 5.8–6.

8. After phase A and phase B are mixed, the overall pH is controlled at 6.5–7.

2.

9. The method for preparing the injectable gel for constructing an osteomyelitis model according to claim 1, characterized in that, The Sau-DualGel precursor described in S5 contains 5–15 mg / mL of Sau-Sr / Mg-CaP-MC microcavities.

10. An injectable gel for constructing an osteomyelitis model, characterized in that, It is prepared using the method for preparing an injectable gel for constructing an osteomyelitis model as described in any one of claims 1 to 9.

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