An injectable hydrogel for alveolar bone repair and its preparation method and application

Abstract

The application discloses an injectable hydrogel for alveolar bone repair, which comprises a first component and a second component, wherein the first component comprises 4-carboxyl-3-fluorophenylboronic acid modified carboxymethyl chitosan CMCS-FPBA and beta-tricalcium phosphate, and the second component comprises oxidized dextran, PPS-HA nanomicelles and curcumin. The application further discloses a preparation method and application of the injectable hydrogel. The injectable hydrogel has the effects of antioxidation, anti-inflammation and bone tissue regeneration promotion, has high biocompatibility, and can be customized, and can be used for alveolar bone repair.

Description

Technical Field

[0001] This invention belongs to the field of bone regeneration and biological repair, specifically relating to an injectable hydrogel for alveolar bone repair, its preparation method, and its application. Background Technology

[0002] Alveolar bone is the skeletal structure to which the roots of teeth are attached. Located in the maxilla and mandible within the oral cavity, it is a crucial structure for supporting and stabilizing teeth. The primary function of alveolar bone is to support teeth and provide sufficient stability for functions such as chewing. Alveolar bone defects refer to the loss or absence of alveolar bone due to various causes (such as periodontal disease, trauma, tooth extraction, infection, etc.). Alveolar bone defects not only affect tooth stability but can also lead to tooth loosening, loss, and a decline in oral function. Current treatment methods for alveolar bone defects include medication and oral care, bone grafting, guided bone regeneration (GBR), dental implants, and biomaterials.

[0003] While infection can be controlled in basic periodontal treatment, its effectiveness is limited for severe bone defects. Traditional pharmacological methods for regulating bone metabolism, such as bisphosphonates, can help inhibit bone resorption, but they may carry the risk of osteonecrosis of the jaw. Furthermore, autologous bone grafting is considered the gold standard for periodontal bone repair, but its clinical application is greatly limited due to donor site morbidity, limited bone resources, and surgical complexity. Allogeneic or xenogeneic bone, while alternatives, may face risks of immune rejection or disease transmission, and their resorption rates are high. In GBR (guided bone regeneration) techniques, the barrier membrane may shift or be exposed, leading to infection or surgical failure. Bone tissue engineering, as an emerging therapeutic approach, shows promising repair potential through the synergistic effects of combining biomaterials, cells, and growth factors. However, the high cost and low cell viability of growth factors limit their widespread application. Therefore, researchers are working to develop alternative biomaterials, especially hydrogels based on natural ingredients. Hydrogels, due to their excellent biocompatibility and ability to mimic the extracellular matrix, show great potential in bone tissue regeneration.

[0004] However, traditional hydrogels are difficult to completely fill irregular periodontal defects, which may lead to tissue damage and prolong surgical time. In contrast, injectable hydrogels, due to their good flexibility and ease of handling, have become a potential material for solving this problem and are becoming a research hotspot in the field of alveolar bone repair. Summary of the Invention

[0005] This invention aims to solve the following technical problems:

[0006] (1) Poor surgical complexity and adaptability: Traditional bone grafting requires open surgery, which is highly invasive; pre-shaped scaffolds are difficult to fit complex defects. The CMCS-FPBA / ODex dynamic network allows the gel to be injected through a needle and to self-repair and shape in vivo, adapting to irregular bone defects.

[0007] (2) Mismatch between degradation rate and bone regeneration: Single materials (such as pure β-TCP) degrade too quickly, or synthetic polymers degrade too slowly, resulting in asynchronous bone growth. Multi-stage responsive degradation occurs, with β-TCP providing early calcium and phosphorus ion release to promote mineralization. PPS-HA, through oxidative responsive degradation via thioether bonds, matches the post-inflammatory repair needs. ODex's pH-sensitive degradation adapts to changes in the oral microenvironment, achieving time-sequential degradation.

[0008] (3) Insufficient mechanical properties of traditional materials: Traditional β-TCP is brittle and easily breaks (such as failure under biting force). By forming a dynamic cross-linked network with CMCS-FPBA and ODex, β-TCP particles are encapsulated, stress is dispersed and compressive strength is improved, avoiding the brittle defects of pure β-TCP, and combining toughness and strength.

[0009] (4) Insufficient bioactivity and low alveolar bone integration efficiency: Free Cur has a short half-life and insufficient local concentration; traditional scaffolds only provide passive support and lack signals to actively induce bone regeneration. By utilizing the oxidative responsiveness of PPS (increased ROS at the site of inflammation) and the enzymatic responsiveness of HA (enrichment of hyaluronidase), targeted sustained release of Cur can be achieved, thus prolonging the therapeutic effect.

[0010] (5) Infection and inflammation control: The oral microbiota is complex, and graft exposure can easily lead to infection. Traditional antibiotics are prone to resistance. CMCS antibacterial + Cur anti-inflammatory + PPS-HA responsive drug delivery system. Accelerates the release of antibacterial components in the infection microenvironment (acidic, high ROS), enabling precise regulation.

[0011] (6) Personalized treatment and long-term stability: Individual differences among patients affect the bone regeneration effect. By adjusting the cross-linking density, different mechanical load requirements can be matched (such as posterior teeth region and anterior teeth region).

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides an injectable hydrogel for alveolar bone repair, comprising a first component and a second component, wherein the first component comprises 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan (CMCS-FPBA) and β-tricalcium phosphate (β-TCP), and the second component comprises oxidized dextran (ODex), PPS-HA nanomicelles and curcumin (Cur).

[0014] Preferably, the injectable hydrogel comprises, by mass-volume percentage, 0.2-2% of 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 0.5-1.5% of β-tricalcium phosphate, 1-5% of oxidized dextran, 0.2-1% of PPS-HA nanomicelles and 0.1-5% of curcumin.

[0015] More preferably, by mass-volume percentage, the injectable hydrogel comprises 0.35-1% of 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 0.5-1% of β-tricalcium phosphate, 2-4% of oxidized dextran, 0.5-1% of PPS-HA nanomicelles and 0.5-1% of curcumin.

[0016] Most preferably, the injectable hydrogel comprises, by weight-volume percentage, 0.7% 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 1% β-tricalcium phosphate, 4% oxidized dextran, 1% PPS-HA nanomicelles and 0.5% curcumin.

[0017] Preferably, the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan is prepared by the following steps:

[0018] Carboxymethyl chitosan was dissolved in PBS to obtain a carboxymethyl chitosan solution. Then, FPBA, EDC, and NHS were dissolved in DMSO and stirred to activate the carboxyl groups to obtain an FPBA solution. The carboxymethyl chitosan solution was added to the FPBA solution, and the pH was adjusted to 12. The mixture was stirred overnight. After the reaction was complete, the solution was dialyzed. After dialysis, the supernatant was filtered and lyophilized to obtain the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan.

[0019] More preferably, the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan is prepared by the following steps: 1 g of carboxymethyl chitosan is dissolved in 1000 mL of PBS to obtain a carboxymethyl chitosan solution; then, 2.75 g of FPBA, 4.125 g of EDC, and 2.75 g of NHS are dissolved in 100 mL of DMSO and stirred for 4 h to activate the carboxyl groups to obtain an FPBA solution; the carboxymethyl chitosan solution is added to the FPBA solution, and the pH is adjusted to 12, and the reaction is stirred overnight; after the reaction is complete, the solution is dialyzed using an 8000 molecular weight dialysis bag; after dialysis, the supernatant is filtered and lyophilized to obtain the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan.

[0020] Preferably, the oxidized glucose is prepared by the following steps:

[0021] Weigh out dextran and dissolve it in water. After dissolving in a water bath, cool to room temperature, add sodium periodate, react in the dark, add ethylene glycol to terminate the reaction, continue stirring, dialyze, and freeze dry to collect.

[0022] More preferably, the oxidized glucose is prepared by the following steps: 5 g of dextran is dissolved in 250 mL of water, dissolved in a water bath at 60°C, cooled to room temperature, 5 g of sodium periodate is added, and the reaction is carried out in the dark for 3.5 h. 3 mL of ethylene glycol is added to terminate the reaction, and stirring is continued for 1 h. The mixture is dialyzed using an 8000 dialysis bag and collected by freeze-drying.

[0023] Preferably, the PPS-HA nanomicelles are prepared by the following steps:

[0024] Under ice bath conditions, 3-mercaptopropionic acid was added to anhydrous tetrahydrofuran and mixed with magnetic stirring; 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the reaction mixture was stirred under a nitrogen atmosphere; then, propylene sulfide was added dropwise, and the reaction mixture was stirred overnight; subsequently, the reaction was quenched by adding H2O, purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain yellow oily PPS; PPS was added to dichloromethane and dissolved with magnetic stirring; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added, and the mixture was stirred until dissolved at room temperature; then, ethylenediamine was added dropwise to the mixture, and the mixture was stirred overnight at room temperature; subsequently, dichloromethane was added to dilute the reaction solution, and the mixture was washed successively with H2O and saturated NaCl, dried over MgSO4, and filtered. The solution was concentrated under reduced pressure to obtain PPS-NH2, which was then dried for later use. Sodium hyaluronate was dialyzed overnight in HCl solution and then lyophilized to obtain acidic hyaluronic acid. H2O and acidic hyaluronic acid were added and magnetically stirred to dissolve. Then N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the mixture was stirred at room temperature until completely dissolved to obtain PPS-NH2. PPS-NH2 was dissolved in tetrahydrofuran and added dropwise to the reaction system. The reaction was continued under stirring at room temperature and N2 protection. The mixture was dialyzed three times, lyophilized to remove the solvent, and the PPS-HA nanomicelles were obtained.

[0025] More preferably, the PPS-HA nanomicelles are prepared by the following steps: 1.15 mmol of 3-mercaptopropionic acid (3-MPA) is added to 30 mL of anhydrous tetrahydrofuran under ice bath conditions, and the mixture is stirred magnetically; 3.45 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene is added, and the reaction mixture is stirred for 30 min under a nitrogen atmosphere. Then, 21.15 mmol of propylene sulfide was added dropwise, and the reaction mixture was stirred overnight at 60 °C. The reaction was then quenched by adding 5 mL of H₂O, purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain a yellow oily PPS. 100 μmol of dried PPS was added to 20 mL of dichloromethane and dissolved by magnetic stirring. 200 μmol of N-hydroxysuccinimide (NHS) and 250 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) were added, and the mixture was stirred at room temperature for 30 min until dissolved. Then, 2 mmol of ethylenediamine was added dropwise to the mixture, and the mixture was stirred overnight at room temperature. Finally, 20 mL of dichloromethane was added to dilute the reaction solution, which was washed successively with H₂O and saturated NaCl, dried over MgSO₄, and filtered. The solution was concentrated under reduced pressure to obtain PPS-NH2, which was then dried for later use. Sodium hyaluronate was dialyzed overnight in 0.01 M HCl solution and then lyophilized to obtain acidic hyaluronic acid (HA). 10 mL of H2O and 100 mg of acidic HA were added to a 50 mL beaker and magnetically stirred to dissolve. Then, 60 μmol of N-hydroxysuccinimide and 75 μmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the mixture was stirred at room temperature for 30 min until completely dissolved to obtain PPS-NH2. 40 mg of PPS-NH2 was dissolved in 1 mL of tetrahydrofuran and added dropwise to the reaction system. The reaction was continued to be stirred for 24 hours at room temperature under N2 protection. The mixture was dialyzed three times with water / methanol at a 1:1 ratio for one day, and then dialyzed three times with distilled water for one day. The solvent was removed by lyophilization to obtain the PPS-HA nanomicelles.

[0026] Secondly, the present invention also provides a method for preparing the injectable hydrogel for alveolar bone repair, comprising the following steps:

[0027] S1. Preparation of PPS-HA@Cur nanomicelles;

[0028] S2. Preparation of CMCS-FPBA@β-TCP / ODex@PPS-HA@Cur hydrogel.

[0029] Step S1 includes: dissolving PPS-HA nanomicelles in PBS; dissolving curcumin in acetone / ethanol solution; adding the organic phase liquid to the aqueous phase under stirring, rotary evaporating for 3-5 min, sonicating at 40 ℃ and 45 rpm for 10 min, and filtering through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0030] Step S2 includes: adding CMCS-FPBA to deionized water to obtain a CMCS-FPBA solution; adding β-TCP to 2.5 mL of the CMCS-FPBA solution and mixing thoroughly to obtain solution A; adding oxidized glucose ODex to PPS-HA@Cur nanomicelles and mixing thoroughly to obtain solution B; mixing solution A and solution B thoroughly and adjusting the pH to 7-8 to obtain the injectable hydrogel.

[0031] Thirdly, the present invention also provides the application of the injectable hydrogel for alveolar bone repair in the preparation of antioxidant, anti-inflammatory and bone tissue regeneration promoting drugs.

[0032] Fourthly, the present invention also provides the application of the injectable hydrogel for alveolar bone repair in the preparation of alveolar bone repair drugs.

[0033] Compared with the prior art, the advantages of the technical solution of the present invention are as follows:

[0034] (1) Design of dynamic self-healing crosslinking network: Dynamic crosslinking of CMCS-FPBA with ODex via borate ester bonds. By modifying phenylboronic acid (FPBA) with carboxymethyl chitosan (CMCS), reversible borate ester bonds are formed with the vicinal diol groups in oxidized dextran (ODex), which endows the hydrogel with shear thinning (fluidity during injection) and self-healing ability (restoring structural integrity in vivo), thereby improving compressive strength and achieving self-healing.

[0035] (2) Synergistic mechanism of multi-level responsive degradation and drug controlled release system: pH / ROS / enzyme three-response linkage to match the needs of different stages of bone regeneration (early anti-infection, mid-term bone promotion, late-term vascularization).

[0036] (3) β-TCP+HA+Cur multi-signal synergy, simultaneously promotes bone formation, inhibits bone resorption, and promotes angiogenesis.

[0037] (4) CMCS antibacterial + Cur anti-inflammatory + targeted drug delivery, doubly reducing the risk of infection.

[0038] (5) It can be injected with minimally invasive techniques and self-repairing molding, adapting to complex defects and promoting minimally invasive alveolar bone regeneration.

[0039] (6) Adjustable cross-linking density + pathological microenvironment response, adapting to the needs of different patients.

[0040] (7) Personalized customization: By adjusting the CMCS / ODex ratio, it can be adapted to different mechanical load areas (such as low load on anterior teeth and high load on posterior teeth).

[0041] (8) Good biocompatibility, safe and without side effects. Attached Figure Description

[0042] Figure 1 An injectable image of the hydrogel is shown.

[0043] Figure 2 An image of the hydrogel's self-healing mechanism is shown.

[0044] Figure 3 The cumulative release rate of curcumin is shown.

[0045] Figure 4 The anti-inflammatory effect of the hydrogel was demonstrated. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] The preparation method of hydrogel includes the following steps:

[0048] (1) Preparation of CMCS-FPBA:

[0049] 1 g of CMCS was dissolved in 1000 mL of PBS. Then, 2.75 g of FPBA, 4.125 g of EDC, and 2.75 g of NHS were dissolved in 100 mL of DMSO, and the mixture was stirred for 4 h to activate the carboxyl groups. The CMCS solution was then added to the FPBA solution, and the pH was adjusted to 12. The mixture was stirred overnight. After the reaction was complete, the solution was dialyzed using an 8000 molecular weight dialysis bag. After dialysis, the supernatant was collected, lyophilized, and the CMCS-FPBA was obtained.

[0050] (2) Preparation of ODex:

[0051] Weigh 5 g of dextran and dissolve it in 250 mL of water. After dissolving in a water bath at 60 °C, cool to room temperature, add 5 g of sodium periodate, and react in the dark for 3.5 h. Add 3 mL of ethylene glycol to terminate the reaction, continue stirring for 1 h, dialyze through an 8000 dialysis bag, and collect by freeze-drying.

[0052] (3) Preparation of PPS-HA nanomicelles:

[0053] Under ice bath conditions, 1.15 mmol of 3-mercaptopropionic acid (3-MPA) was added to 30 mL of anhydrous tetrahydrofuran and mixed with magnetic stirring. Then, 3.45 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the reaction mixture was stirred for 30 min under a nitrogen atmosphere. Next, 21.15 mmol of propylene sulfide was added dropwise, and the reaction mixture was stirred overnight at 60 °C. Afterward, the reaction was quenched by adding 5 mL of H₂O, purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain a yellow oily PPS. 100 μmol of dried PPS was added to 20 mL of dichloromethane and dissolved with magnetic stirring. Then, 200 μmol of N-hydroxysuccinimide (NHS) and 250 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) were added, and the mixture was stirred at room temperature for 30 min until dissolved. Then, 2 mmol of ethylenediamine was added dropwise to the mixture, and stirring was continued overnight at room temperature. Afterward, 20 mL of dichloromethane was added to dilute the reaction solution, followed by washing with H2O and saturated NaCl, drying with MgSO4, and filtering. The solution was concentrated under reduced pressure to obtain PPS-NH2, which was then dried for later use. Sodium hyaluronate was dialyzed overnight in 0.01 M HCl solution and then lyophilized to obtain acidic hyaluronic acid (HA). 10 mL of H2O and 100 mg of acidic HA were added to a 50 mL beaker and dissolved by magnetic stirring. Then, 60 μmol of N-hydroxysuccinimide and 75 μmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the mixture was stirred at room temperature for 30 min until completely dissolved to obtain PPS-NH2. 2。 40 mg of PPS-NH2 was dissolved in 1 mL of tetrahydrofuran and added dropwise to the reaction system. The reaction was continued with stirring for 24 hours at room temperature under N2 protection. The mixture was dialyzed three times with water / methanol at a 1:1 ratio, with each dialyzing lasting one day. Then, it was dialyzed three times with distilled water, with each dialyzing lasting one day. The solvent was removed by lyophilization to obtain the PPS-HA nanomicelles.

[0054] (4) Preparation of PPS-HA@Cur nanomicelles:

[0055] A certain amount of PPS-HA was dissolved in 10 mL of PBS; a certain amount of Cur was dissolved in 2 mL of acetone / ethanol solution (volume ratio 3:2); the organic phase liquid was added to the aqueous phase (added under stirring, using a rotary evaporator), rotary evaporated for 3-5 min (40℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0056] (5) Preparation of CMCS-FPBA@β-TCP / ODex@PPS-HA@Cur hydrogel

[0057] A certain amount of CMCS-FPBA was added to 10 mL of deionized water to obtain a CMCS-FPBA solution. A certain amount of β-TCP was added to 2.5 mL of the CMCS-FPBA solution and mixed evenly to obtain solution A. A certain amount of ODex was added to PPS-HA@Cur nanomicelles and the solution was mixed evenly to obtain solution B. Solution A and solution B were mixed and the pH was adjusted to 7-8 to finally form a microgel assembly.

[0058] Example 1:

[0059] Add 0.007g of CMCS-FPBA to 10mL of deionized water to obtain a 0.7% CMCS-FPBA solution. Add 0.025g of β-TCP to 2.5mL of the 0.7% CMCS-FPBA solution and mix well to obtain solution A.

[0060] 0.01 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.01 g of curcumin (Cur) was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0061] Take 0.1 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0062] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0063] Example 2:

[0064] Add 0.007g of CMCS-FPBA to 10mL of deionized water to obtain a 0.7% CMCS-FPBA solution. Add 0.05g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0065] 0.02 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.02 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0066] Take 0.1 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0067] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0068] Example 3:

[0069] Add 0.007g of CMCS-FPBA to 10mL of deionized water to obtain a 0.7% CMCS-FPBA solution. Add 0.025g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0070] 0.01 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.01 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0071] Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0072] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0073] Example 4:

[0074] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 0.025g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0075] 0.01 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.02 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0076] Take 0.1 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0077] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0078] Example 5:

[0079] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 0.05g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0080] 0.02 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.01 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0081] Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0082] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0083] Example 6:

[0084] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 0.05g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0085] 0.02 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.02 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0086] Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0087] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0088] Example 7:

[0089] Add 0.02g of CMCS-FPBA to 10mL of deionized water to obtain a 2% CMCS-FPBA solution. Add 0.05g of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0090] 0.02 g of PPS-HA nanomicelles were dissolved in 10 mL of PBS; 0.02 g of Cur was dissolved in 2 mL of acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporated for 3-5 min (40 ℃, 45 rpm), sonicated for 10 min, and filtered through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles.

[0091] Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles. Mix the solution evenly to obtain solution B.

[0092] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0093] Comparative Example 1:

[0094] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 2mg of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0095] Add 0.2g of ODex to solution A, adjust the pH to 7-8, and finally form microgel assemblies.

[0096] Comparative Example 2:

[0097] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 2mg of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0098] Dissolve 0.02 g of PPS-HA in 10 mL of PBS to obtain PPS-HA nanomicelles. Add 0.2 g of ODex to 2.5 mL of PPS-HA nanomicelles and mix the solutions thoroughly to obtain solution B.

[0099] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0100] Comparative Example 3:

[0101] Add 0.014g of CMCS-FPBA to 10mL of deionized water to obtain a 1.4% CMCS-FPBA solution. Add 2mg of β-TCP to 2.5mL of CMCS-FPBA solution and mix well to obtain solution A.

[0102] Dissolve 0.02 g of Cur in 2.5 mL of a mixed solution of DMSO and water (1:4), add 0.2 g of ODex, mix well, and obtain solution B.

[0103] Mix solution A and solution B, adjust the pH to 7-8, and finally form microgel assemblies.

[0104] 1. Hydrogel Injectable Images

[0105] Figure 1 An injectable image of the hydrogel of Example 6 is shown.

[0106] 2. Images of hydrogel self-healing

[0107] Figure 2 An image of the self-healing hydrogel of Example 6 is shown.

[0108] 3. Viscosity test

[0109] Test method: The hydrogel was injected onto the test sample stage of the rotational rheometer. The test temperature was set to 37 °C and the shear frequency to 1 Hz. The viscosity of the liquid was tested using a stainless steel cone plate. The results are shown in Table 1.

[0110] Table 1. Viscosity test results

[0111]

[0112] Results: As shown in Table 1, the viscosity of the hydrogel is affected by the ratio of CMCS-FPBA to ODex. When the CMCS-FPBA content is 0.7% and the ODex content is 4%, the hydrogel viscosity is 66.15 Pa / s, which ensures sufficient adhesion between the hydrogel and bone tissue, prevents hydrogel detachment or displacement, and ensures its continuous function during the repair process.

[0113] However, hydrogels with excessively low viscosity may flow too quickly in vivo, affecting their persistence and drug release control. Hydrogels with excessively high viscosity (as in Example 7) may clog the injection needle site, leading to injection difficulties. In Example 6, the hydrogel synthesized with a ratio of 0.7% CMCS-FPBA and 4% ODex showed the smoothest injection. Figure 1 and Figure 2 These are schematic images of hydrogel injection and self-healing in Example 6. Other hydrogels in other examples also exhibit a similar appearance.

[0114] 4. Degradation performance test

[0115] Test method: The prepared microgel assemblies were first freeze-dried at -80 °C. The initial weight of the freeze-dried hydrogel (the gelatin sponge was kept consistent with this initial weight) was recorded as W0. The prepared freeze-dried hydrogel samples were placed in 24-well plates, and a fixed volume of collagenase solution was added using a pipette to completely submerge the hydrogel samples. The plates were then placed in a 37 °C constant temperature shaker. After 12 hours, the samples were removed, excess degradation solution was discarded, the samples were freeze-dried, weighed, and the weight was recorded as W. t The degradation rate is calculated using the following formula:

[0116]

[0117] Table 2. Degradation performance analysis

[0118]

[0119] Results: As shown in Table 2, the degradation rate of the hydrogel in the collagenase degradation solution of Example 6 of the present invention was 40.14±2.04% after 12 hours, indicating that the hydrogel of the present invention has good stability and anti-degradation properties and can better adapt to the complex oral environment.

[0120] Curcumin release

[0121] Test Method: First, standard curves were established using different concentrations of curcumin (500, 250, 125, 62.5, 31.75 μg / mL). Then, H2O2 solutions of different concentrations (0 mM, 0.25 mM, 0.5 mM) were prepared. 400 μL of the microgel from Example 6 was placed in 1 mL of H2O2 solutions of specific concentrations (0.25 mM, 0.50 mM) and incubated at 37 ℃ and 100 rpm in a constant temperature shaking incubator. Samples were taken at different time points (1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 192 h), with 1 mL of culture medium at different H2O2 concentrations, and replaced with an equal volume of fresh PBS. The samples were centrifuged at 4000 rpm for 5 min. The absorbance of curcumin in PBS solution was measured using UV spectrophotometry at an excitation wavelength of 425 nm.

[0122] Calculation formula: Cumulative release rate (%) = (Cumulative drug release amount / Drug dosage) × 100%

[0123] Figure 3 The cumulative release rate of curcumin is shown.

[0124] Results indicate: From Figure 3It was found that curcumin release from PBS was much slower than from H2O2, with a cumulative release of 49.4% after 192 h. However, cultivation in 0.25 mM H2O2 solution increased the curcumin release rate, with a cumulative release of 69.3% after 192 h, demonstrating high sensitivity to H2O2. In 0.50 mM H2O2 solution, the cumulative release after 192 h was 88.9%, significantly increasing the curcumin release rate. This indicates that the concentration of H2O2 solution can accelerate the release of curcumin. This may be because PPS-HA nanomicelles underwent an oxidation reaction in a ROS environment, leading to polymer chain breakage or destruction of cross-linking structures. This reaction causes changes in the micelle structure, resulting in faster release of curcumin from PPS-HA nanomicelles, reflecting its high adaptability to environmental changes. This ROS-responsive release mechanism allows the drug to be released at specific times and locations. More importantly, this precise control of the drug release rate by adjusting the concentration of ROS in the external environment can effectively improve the targeting and therapeutic efficacy of curcumin.

[0125] 6. Antioxidant capacity test

[0126] Test method: The antioxidant properties of different examples and comparative examples were evaluated by using the method of scavenging 1,1-diphenyl-2-pyridylhydrazide (DPPH) free radicals.

[0127] The mixtures of different examples and comparative samples with DPPH reagent were incubated in the dark with stirring for 30 minutes, and the remaining DPPH was analyzed by UV-Vis spectroscopy. The formula for determining the DPPH scavenging rate is:

[0128] D (%) = [[A 空白 - (A 测定 -A 对照 )] / A 空白 ]×100%

[0129] Table 3. DPPH free radical scavenging rate (%)

[0130]

[0131] Results: Table 3 shows that PPS-HA-containing nanomicelles exhibit free radical scavenging activity. This is likely because the sulfur atoms in PPS-HA are readily oxidized by ROS to form sulfoxides, which are further oxidized to sulfones. Compared to smaller antioxidant molecules, PPS polymers contain multiple ROS reaction sites, enabling them to scavenge more ROS. Furthermore, compared to Cur hydrogels alone, Cur-containing PPS-HA nanomicelles demonstrate higher free radical scavenging efficiency. This is because the phenolic hydroxyl groups in Cur are strong free radical terminators; these hydroxyl groups can donate electrons to free radicals, thereby reducing them to form stable molecules. This indicates that hydrogels containing both Cur and PPS-HA have higher free radical scavenging capacity, suggesting a synergistic antioxidant effect between Cur and PPS-HA.

[0132] 7. In vitro antibacterial test

[0133] Test method: Porphyromonas gingivalis, Escherichia coli, and Staphylococcus aureus were revived and cultured to the logarithmic growth phase. After centrifugation, the bacteria were collected and resuspended in physiological saline. The final bacterial concentration was 1×10⁻⁶. 8 CFU / mL. Add the corresponding hydrogel sample to a 24-well plate. Add 500 μL of physiological saline to the control group. Then add 100 μL of diluted bacterial suspension and 1 mL of sterile physiological saline to each well, incubate at 37 ℃ for 24 hours, serially dilute and spread on BA medium or LB agar plates, and count.

[0134] Table 4. In vitro antibacterial rate (%)

[0135]

[0136] The results show that, as indicated in Table 4, the addition of PPS-HA enhances the antibacterial properties of curcumin. This is because PPS-HA improves the solubility of curcumin, which helps curcumin exert its antibacterial effect more effectively. Furthermore, the slow release of curcumin in the PPS-HA composite material helps maintain its antibacterial activity for a longer period. This not only increases the local concentration of curcumin but also prolongs its action time at the lesion site, thereby improving the antibacterial effect. This suggests that the simultaneous presence of curcumin and PPS-HA may produce a synergistic effect, enhancing the overall antibacterial activity.

[0137] 8. Anti-inflammatory evaluation:

[0138] Test Method: SD rats were weighed and anesthetized with 2% sodium pentobarbital via intraperitoneal injection. The rats were then fixed in a supine position on a rat board, and the mandible was pulled to expose the maxillary dentition as fully as possible. The mesial gingiva of the first maxillary molar was incised with a scalpel, the gingival flap was gently flipped open, hemostasis was achieved with cotton balls, and the bone surface was exposed. A 2mm diameter ball bur was used to remove the mesial alveolar bone of the mesial root of the first molar until the bur was completely inserted to determine the cavity depth. A 2mm diameter fissure bur was then used to refine the cavity shape. Water was continuously sprayed during the procedure to prevent bone necrosis. After implanting the appropriate materials according to the group, the gingiva was sutured. Postoperatively, penicillin was administered intramuscularly to prevent infection. Ten days post-surgery, alveolar bone tissue samples were collected, washed with pre-chilled PBS (0.02 mol / L, pH 7.0-7.2) to remove blood, and weighed for later use. 1.0 g of tissue block was transferred to a glass homogenizer, and 5 mL of pre-chilled PBS was added for thorough homogenization. This process should be performed on ice. The resulting homogenate was repeatedly frozen and thawed twice. The prepared homogenate was centrifuged at 5000×g for 5 minutes, and the supernatant was collected for detection using an ELISA kit.

[0139] Figure 4 The anti-inflammatory effect of the hydrogel was demonstrated.

[0140] Results indicate: From Figure 4 It can be seen that, compared with the saline group and Comparative Example 3, the hydrogel scaffold of Example 6 can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6 and IL-1β, and provide a stable microenvironment for alveolar bone tissue.

[0141] In summary, the hydrogel of this invention uses CMCS-FPBA and ODex as matrix materials, and introduces Cur and PPS-HA to endow the hydrogel with antibacterial, antioxidant and immunomodulatory functions. By optimizing the proportion of each component, the hydrogel obtained has good injectability, stability and anti-degradation properties, enabling it to better play its role in repairing alveolar bone in the complex oral environment.

Claims

1. An injectable hydrogel for alveolar bone repair, characterized in that, The product comprises a first component and a second component. The first component comprises 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan (CMCS-FPBA) and β-tricalcium phosphate (β-TCP). The second component comprises oxidized dextran (ODex), PPS-HA nanomicelles, and curcumin (Cur). The PPS-HA nanomicelles and curcumin (Cur) are used to prepare PPS-HA@Cur nanomicelles. The 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan was prepared by the following steps: Carboxymethyl chitosan was dissolved in PBS to obtain a carboxymethyl chitosan solution. Then, 4-carboxy-3-fluorophenylboronic acid, EDC, and NHS were dissolved in DMSO and stirred to activate the carboxyl groups, yielding a 4-carboxy-3-fluorophenylboronic acid solution. The carboxymethyl chitosan solution was added to the 4-carboxy-3-fluorophenylboronic acid solution, and the pH was adjusted to 12. The mixture was stirred overnight. After the reaction was complete, the solution was dialyzed. After dialysis, the supernatant was collected by filtration and lyophilized to obtain the 4-carboxy-3-fluorophenylboronic acid-modified carboxymethyl chitosan CMCS-FPBA. The PPS-HA nanomicelles were prepared by the following steps: Under ice bath conditions, 3-mercaptopropionic acid was added to anhydrous tetrahydrofuran and mixed with magnetic stirring; 1,8-diazabicyclo[5.4.0]undec-7-ene was added, and the reaction mixture was stirred under a nitrogen atmosphere; then, propylene sulfide was added dropwise, and the reaction mixture was stirred overnight; subsequently, the reaction was quenched by adding H2O, purified by precipitation in cold methanol, and the solvent was evaporated under reduced pressure to obtain yellow oily PPS; PPS was added to dichloromethane and dissolved with magnetic stirring; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added, and the mixture was stirred until dissolved at room temperature; then, ethylenediamine was added dropwise to the mixture, and the mixture was stirred overnight at room temperature; subsequently, dichloromethane was added... The reaction solution was diluted with methane, washed successively with H2O and saturated NaCl, dried with MgSO4, and filtered. The solution was concentrated under reduced pressure to obtain PPS-NH2, which was then dried for later use. Sodium hyaluronate was dialyzed overnight in HCl solution and then lyophilized to obtain acidic hyaluronic acid. H2O and acidic hyaluronic acid were added and magnetically stirred to dissolve. Then, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the mixture was stirred at room temperature until completely dissolved. PPS-NH2 was dissolved in tetrahydrofuran and added dropwise to the reaction system. The reaction was continued under stirring at room temperature and N2 protection. The mixture was dialyzed three times, lyophilized to remove the solvent, and the PPS-HA nanomicelles were obtained.

2. The injectable hydrogel according to claim 1, characterized in that, The injectable hydrogel comprises, by weight-volume percentage, 0.2-2% of 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 0.5-1.5% of β-tricalcium phosphate, 1-5% of oxidized dextran, 0.2-1% of PPS-HA nanomicelles, and 0.1-5% of curcumin.

3. The injectable hydrogel according to claim 1, characterized in that, The injectable hydrogel comprises, by weight-volume percentage, 0.35-1% of 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 0.5-1% of β-tricalcium phosphate, 2-4% of oxidized dextran, 0.5-1% of PPS-HA nanomicelles and 0.5-1% of curcumin.

4. The injectable hydrogel according to claim 1, characterized in that, The injectable hydrogel comprises, by weight-volume percentage, 0.7% 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan, 1% β-tricalcium phosphate, 4% oxidized dextran, 1% PPS-HA nanomicelles and 0.5% curcumin.

5. The injectable hydrogel according to any one of claims 1 to 4, characterized in that, The oxidized dextran was prepared by the following steps: Weigh out dextran and dissolve it in water. After dissolving in a water bath, cool to room temperature, add sodium periodate, and react in the dark. Add ethylene glycol to terminate the reaction, continue stirring, dialyze, and freeze dry to collect the product.

6. A method for preparing an injectable hydrogel as described in any one of claims 1 to 5, characterized in that... Includes the following steps: S1. Preparation of PPS-HA@Cur nanomicelles; S2. Preparation of CMCS-FPBA@β-TCP / ODex@PPS-HA@Cur hydrogel; Step S1 includes: dissolving PPS-HA nanomicelles in PBS to obtain an aqueous phase; dissolving curcumin in acetone and ethanol solutions to obtain an organic phase; adding the organic phase liquid to the aqueous phase under stirring, rotary evaporating for 3-5 min at 40 ℃ and 45 rpm; sonicating for 5-10 min; and filtering through a 0.45 μm microporous membrane to obtain PPS-HA@Cur nanomicelles. Step S2 includes: adding CMCS-FPBA to deionized water to obtain a CMCS-FPBA solution; adding β-TCP to the CMCS-FPBA solution and mixing them evenly to obtain solution A; adding oxidized dextran ODex to PPS-HA@Cur nanomicelles and mixing them evenly to obtain solution B; mixing solutions A and B evenly and adjusting the pH to 7-8 to obtain the injectable hydrogel.

7. The use of the injectable hydrogel as described in any one of claims 1 to 5 in the preparation of alveolar bone repair drugs.

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