Injectable hydrogel for repairing alveolar bone as well as preparation method and application of injectable hydrogel

Abstract

The invention discloses injectable hydrogel for alveolar bone repair, the injectable hydrogel comprises a first component and a second component, 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 nano-micelle and curcumin. The invention also discloses a preparation method and application of the injectable hydrogel. The injectable hydrogel disclosed by the invention has the effects of resisting oxidation, resisting inflammation and promoting bone tissue regeneration, is high in biocompatibility, can be customized in a personalized manner, and can be used for repairing alveolar bones.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bone regeneration and biological repair, and particularly relates to an injectable hydrogel for alveolar bone repair and a preparation method and application thereof. BACKGROUND

[0002] Alveolar bone is the part of the bone that the roots of the teeth are attached to, located in the upper and lower jaws in the oral cavity, and is an important structure for supporting and fixing teeth. The main function of alveolar bone is to support teeth and provide sufficient stability so that teeth can perform functions such as mastication. Alveolar bone defect refers to the loss or absence of alveolar bone due to various reasons such as periodontal disease, trauma, tooth extraction, infection, etc. Alveolar bone defect not only affects the stability of teeth, but also can cause teeth to become loose and fall out, and oral function to decline. Current treatment methods for alveolar bone defect include drugs and oral care, bone transplantation, guided bone regeneration (GBR), tooth implantation, and biomaterials, etc.

[0003] In periodontal basic treatment, although infection can be controlled, the effect on severe bone defects is limited. Traditional drug regulation of bone metabolism methods, such as bisphosphonates, can help inhibit bone resorption, but they may cause the risk of jaw necrosis. In addition, autologous bone transplantation is considered the gold standard for periodontal bone repair, but due to the incidence of donor sites, limited bone resources, and surgical complexity, its clinical application has been greatly limited. Allogeneic or xenogeneic bone can be used as a substitute, but they may face the risk of immune rejection or disease transmission, and have a high absorption rate. In the GBR technique (guided bone regeneration technique), the barrier membrane can be displaced or exposed, leading to infection or surgical failure. Bone tissue engineering, as a new treatment method, shows good repair prospects through the synergistic effect of combining biomaterials, cells, and growth factors. However, the high cost of growth factors and the limitations of low cell viability have affected its widespread application. Therefore, researchers are working to develop alternative biomaterials, especially hydrogels based on natural ingredients. Hydrogels show great potential in bone tissue regeneration due to their excellent biocompatibility and ability to mimic the properties of the extracellular matrix.

[0004] However, traditional hydrogels are difficult to completely fill irregular periodontal defects, which can cause tissue damage and prolong the operation time. In contrast, injectable hydrogels, due to their good flexibility and ease of operation, have become potential materials to solve this problem and are becoming a research hotspot in the field of alveolar bone repair. SUMMARY

[0005] The present application aims to solve the following technical problems: (1) Poor complexity and adaptability of surgery: Traditional bone grafting requires open surgery, which is traumatic; pre-formed scaffolds are difficult to fit complex defects. CMCS-FPBA / ODex dynamic network enables the gel to be injected through a needle and self-repair in the body to adapt to irregular bone defects.

[0006] (2) Mismatch between degradation rate and bone regeneration: Single materials (such as pure β-TCP) degrade too quickly, or synthetic polymers degrade too slowly, out of sync with bone growth. Multi-stage responsive degradation, in which β-TCP provides early calcium and phosphate ion release to promote mineralization. PPS-HA responds to oxidative degradation through thioether bonds, matching the repair needs in the later stages of inflammation. ODex's pH-sensitive degradation adapts to changes in the oral microenvironment, achieving timed degradation.

[0007] (3) Insufficient mechanical properties of traditional materials: Traditional β-TCP is highly brittle and easily fractured (such as failure under occlusal 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 brittleness of pure β-TCP and combining toughness and strength.

[0008] (4) Insufficient bioactivity and low efficiency of alveolar bone integration: Free Cur has a short half-life and insufficient local concentration; traditional scaffolds only provide passive support and lack active signals to induce bone regeneration. By utilizing the oxidative responsiveness of PPS (increased ROS at inflammatory sites) and the enzyme responsiveness of HA (rich in hyaluronidase), targeted and sustained release of Cur is achieved, prolonging its effectiveness.

[0009] (5) Infection and inflammation control: Oral flora is complex, and exposure of grafts can easily lead to infection, with traditional antibiotics prone to drug resistance. CMCS antibacterial + Cur anti-inflammatory + PPS-HA responsive drug release system. In an infected microenvironment (acidic, high ROS), the release of antibacterial components is accelerated, enabling precise control.

[0010] (6) Personalized treatment and long-term stability: Individual differences in patients affect bone regeneration. By adjusting the cross-linking density to match different mechanical load requirements (such as posterior and anterior regions).

[0011] To achieve the above invention purposes, the present application provides the following technical solutions: In a first aspect, the present application provides an injectable hydrogel for alveolar bone repair, comprising a first component and a second component, wherein the first component comprises 4-carboxyl-3-fluorophenylboric 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).

[0012] Preferably, the injectable hydrogel comprises 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan 0.2-2%, beta-tricalcium phosphate 0.5-1.5%, oxidized dextran 1-5%, PPS-HA nanomicelles 0.2-1%, and curcumin 0.1-5% by mass volume percentage.

[0013] More preferably, the injectable hydrogel comprises 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan 0.35-1%, beta-tricalcium phosphate 0.5-1%, oxidized dextran 2-4%, PPS-HA nanomicelles 0.5-1%, and curcumin 0.5-1% by mass volume percentage.

[0014] Most preferably, the injectable hydrogel comprises 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan 0.7%, beta-tricalcium phosphate 1%, oxidized dextran 4%, PPS-HA nanomicelles 1%, and curcumin 0.5% by mass volume percentage.

[0015] Preferably, the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan is prepared by the following steps: Carboxymethyl chitosan is dissolved in PBS to obtain a carboxymethyl chitosan solution; then, FPBA, EDC, and NHS are dissolved in DMSO, and the carboxyl group is activated by stirring 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 post-reaction solution is dialyzed; after dialysis is complete, the supernatant is filtered, and freeze-drying is performed to obtain the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan.

[0016] 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 the carboxyl group is activated by stirring for 4 h 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 post-reaction solution is dialyzed using a 8000 molecular weight dialysis bag; after dialysis is complete, the supernatant is filtered, and freeze-drying is performed to obtain the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan.

[0017] Preferably, the oxidized dextran is prepared by the following steps: A certain amount of dextran is dissolved in water, and after dissolution in a water bath, it is cooled to room temperature. Sodium periodate is added, and the reaction is carried out in the dark. Ethylene glycol is added to terminate the reaction, and stirring is continued. Dialysis and freeze-drying are performed to collect the product.

[0018] More preferably, the oxidized glucose is prepared by the following steps: 5 g dextran is weighed and dissolved in 250 mL water, dissolved in a 60 °C water bath, and then cooled to room temperature; 5 g sodium periodate is added, and the reaction is carried out in the dark for 3.5 h; 3 mL ethylene glycol is added to terminate the reaction, and stirring is continued for 1 h; dialysis is performed using a 8000 dialysis bag; and lyophilization is performed to collect the product.

[0019] Preferably, the PPS-HA nanomicelles are prepared by the following steps: Under ice bath conditions, 3-mercaptopropionic acid is added to anhydrous tetrahydrofuran, and the mixture is stirred magnetically until uniform; 1,8-diazabicyclo[5.4.0]undec-7-ene is added, and the reaction mixture is stirred under a nitrogen atmosphere; then, propylene sulfide is added dropwise, and the reaction mixture is stirred overnight; after that, the reaction is quenched by adding H2O, and the product is purified by precipitation in cold methanol and evaporation of the solvent under reduced pressure to obtain PPS in the form of a yellow oil; PPS is added to dichloromethane, and the mixture is stirred magnetically until dissolved; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are added, and the mixture is stirred at room temperature until dissolved; then, ethylenediamine is added dropwise to the mixture, and stirring is continued at room temperature overnight; after that, the reaction solution is diluted with dichloromethane, washed sequentially with H2O and saturated NaCl, dried over MgSO4, and filtered. The solution is concentrated under reduced pressure to obtain PPS-NH2, which is dried for later use; sodium hyaluronate is dialyzed against an HCl solution overnight, and then lyophilized to obtain hyaluronic acid in acid form; H2O and the acid form of hyaluronic acid are added, and the mixture is stirred magnetically until dissolved; N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added, and the mixture is stirred at room temperature until completely dissolved to obtain PPS-NH2; PPS-NH2 is dissolved in tetrahydrofuran, and the solution is added dropwise to the reaction system; stirring is continued at room temperature under N2 protection; dialysis is performed three times, and the solvent is removed by lyophilization to obtain the PPS-HA nanomicelles.

[0020] 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 until uniform; 3.45 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene is then added, and the reaction mixture is stirred under a nitrogen atmosphere for 30 min. Then, 21.15 mmol of propylene sulfide is added dropwise, and the reaction mixture is stirred at 60°C overnight; after that, the reaction is quenched by adding 5 mL of H2O, purified by precipitation in cold methanol, and the solvent is evaporated under reduced pressure to obtain PPS in the form of a yellow oil; 100 μmol of dried PPS is added to 20 mL of dichloromethane, and the mixture is stirred magnetically until dissolved; 200 μmol of N-hydroxysuccinimide (NHS) and 250 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) are then added, and the mixture is stirred at room temperature for 30 min until dissolved; then, 2 mmol of ethylenediamine is added dropwise to the mixture, and the stirring is continued at room temperature overnight; after that, the reaction solution is diluted with 20 mL of dichloromethane, washed sequentially with H2O and saturated NaCl, dried over MgSO4, and filtered. The solution is concentrated under reduced pressure to obtain PPS-NH2, which is dried for later use; sodium hyaluronate is dialyzed overnight in a 0.01 M HCl solution, and then lyophilized to obtain hyaluronic acid (HA) in the acid form; 10 mL of H2O and 100 mg of the acid form of HA are added to a 50 mL beaker, and the mixture is stirred magnetically until dissolved; then, 60 μmol of N-hydroxysuccinimide and 75 μmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are added, and the mixture is stirred at room temperature for 30 min until completely dissolved to obtain PPS-NH2; 40 mg of PPS-NH2 is dissolved in 1 mL of tetrahydrofuran, and the solution is added dropwise to the reaction system; the stirring is continued at room temperature under N2 protection for 24 hours; the dialysis is performed three times with water / methanol at a ratio of 1:1 for one day, and then three times with distilled water for one day; the solvent is removed by lyophilization to obtain the PPS-HA nanomicelles.

[0021] In a second aspect, the present application also provides a preparation method of the injectable hydrogel for alveolar bone repair, which comprises the following steps: S1, preparing PPS-HA@Cur nanomicelles; S2, preparing CMCS-FPBA@β-TCP / ODex@PPS-HA@Cur hydrogel.

[0022] The step S1 comprises: taking PPS-HA nanomicelles dissolved in PBS; taking curcumin dissolved in an acetone / ethanol solution; adding the organic phase liquid to the aqueous phase under stirring, rotary evaporation for 3-5 min, 40 DEG C, 45 rpm, ultrasonic for 10 min, and 0.45 um microporous filter membrane, to obtain PPS-HA@Cur nanomicelles.

[0023] The step S2 comprises: 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 uniformly mixing to obtain solution A; adding oxidized glucose ODex to the PPS-HA@Cur nanomicelles and uniformly mixing to obtain solution B; uniformly mixing the solution A and the solution B, and adjusting the pH to 7-8 to obtain the injectable hydrogel.

[0024] In a third aspect, the application further provides a use of the injectable hydrogel for alveolar bone repair in preparation of an anti-oxidation, anti-inflammation and bone tissue regeneration promoting drug.

[0025] In a fourth aspect, the application further provides a use of the injectable hydrogel for alveolar bone repair in preparation of an alveolar bone repair drug.

[0026] Compared with the prior art, the technical scheme of the application has the following advantages: (1) Dynamic self-repairing crosslinking network design: CMCS-FPBA and ODex are dynamically crosslinked through borate ester bonds. By modifying phenylboronic acid (FPBA) with carboxymethyl chitosan (CMCS), the reversible borate ester bonds with the vicinal diol groups in oxidized dextran (ODex) are formed, so that the hydrogel is endowed with shear thinning (flowability during injection) and self-repairing ability (restoring structural integrity in vivo), the compression strength is improved, and self-repairing is realized.

[0027] (2) Synergistic mechanism of multi-stage responsive degradation and drug controlled release system: pH / ROS / enzyme three-response linkage, matching the needs of different stages of bone regeneration (early anti-infection, medium-term pro-osteogenesis, and later vascularization).

[0028] (3) β-TCP+HA+Cur multi-signal synergy, synchronous pro-osteogenesis, anti-osteoclast and pro-vascularization.

[0029] (4) CMCS antibacterial + Cur anti-inflammatory + targeted drug release, double reduction of infection risk.

[0030] (5) Injectable minimally invasive + self-repairing shaping, suitable for complex defects, and promoting minimally invasive alveolar bone regeneration.

[0031] (6) Crosslinking density adjustable + pathological microenvironment response, adapting to different patient needs.

[0032] (7) Personalized customization: by adjusting the ratio of CMCS / ODex, adapting to different mechanical load areas (such as low load of anterior teeth, high load of posterior teeth).

[0033] (8) Good biocompatibility, safe, no side effects. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 An injectable image of the hydrogel is shown.

[0035] Figure 2 An image of the self-healing of the hydrogel is shown.

[0036] Figure 3 The cumulative release rate of curcumin is shown.

[0037] Figure 4 The anti-inflammatory effect of the hydrogel is shown. DETAILED DESCRIPTION

[0038] The following will further illustrate the present application in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present application, and are not used to limit the scope of the present application.

[0039] The preparation method of the hydrogel includes the following steps: (1) Preparation of CMCS-FPBA: Dissolve 1 g of CMCS in 1000 mL of PBS. Then, dissolve 2.75 g of FPBA, 4.125 g of EDC, and 2.75 g of NHS in 100 mL of DMSO, and stir the reaction for 4 h to activate the carboxyl group. Add the CMCS solution to the FPBA solution, adjust the pH value to 12, and stir overnight. After the reaction is complete, use a 8000 molecular weight dialysis bag to dialyze the reaction solution. After dialysis, filter the supernatant, freeze-dry, and obtain CMCS-FPBA.

[0040] (2) Preparation of ODex: Weigh 5 g of dextran and dissolve it in 250 mL of water, dissolve it in a 60°C water bath, and then cool it to room temperature. Add 5 g of sodium periodate, avoid light, and react for 3.5 h. Add 3 mL of ethylene glycol to terminate the reaction, continue stirring for 1 h, dialyze with an 8000 dialysis bag, and freeze-dry to collect.

[0041] (3) Preparation of PPS-HA nanomicelles: To 30 mL of anhydrous tetrahydrofuran, 1.15 mmol of 3-mercaptopropionic acid (3-MPA) was added under ice-bath condition, and the mixture was stirred magnetically until homogeneous; 3.45 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the reaction mixture was stirred under nitrogen atmosphere for 30 min. Then, 21.15 mmol of propylene sulfide was added dropwise, and the reaction mixture was stirred at 60 °C overnight; after that, the reaction was quenched by adding 5 mL of H2O, and the product was purified by precipitation in cold methanol and evaporation of the solvent under reduced pressure to obtain PPS as a yellow oil. To 20 mL of dichloromethane, 100 μmol of dried PPS was added, and the mixture was stirred magnetically until dissolution. 200 μmol of N-hydroxysuccinimide (NHS) and 250 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) were then added, and the mixture was stirred at room temperature for 30 min until dissolution. Then, 2 mmol of ethylenediamine was added dropwise to the mixture, and the stirring was continued at room temperature overnight. After that, the reaction solution was diluted with 20 mL of dichloromethane, washed sequentially with H2O and saturated NaCl, dried over MgSO4, and filtered. The solution was concentrated under reduced pressure to obtain PPS-NH2, which was dried for later use. Sodium hyaluronate was dialyzed overnight against 0.01 M HCl solution and then lyophilized to obtain hyaluronic acid (HA) in acid form. To a 50 mL beaker, 10 mL of H2O and 100 mg of HA in acid form were added, and the mixture was stirred magnetically until dissolution. 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 complete dissolution to obtain PPS-NH 2。 PPS-NH2 was dissolved in 1 mL of tetrahydrofuran and added dropwise to the reaction system; the stirring was continued at room temperature under N2 protection for 24 h. Dialysis was performed three times against water / methanol (1:1) for 1 day, and then dialysis was performed three times against distilled water for 1 day; the solvent was removed by lyophilization to obtain the PPS-HA nanomicelles.

[0042] (4) Preparation of PPS-HA@Cur nanomicelles: 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 flask), rotary evaporation was performed for 3-5 min (40 °C, 45 rpm), ultrasonic treatment was performed for 10 min, and 0.45 um microporous filter membrane was used for filtration to obtain PPS-HA@Cur nanomicelles.

[0043] (5) Preparation of CMCS-FPBA@β-TCP / ODex@PPS-HA@Cur hydrogel A certain amount of CMCS-FPBA was added to 10 mL of deionized water to obtain a CMCS-FPBA solution, and a certain amount of β-TCP was added to 2.5 mL of the CMCS-FPBA solution, and mixed uniformly to obtain solution A; a certain amount of ODex was added to the PPS-HA@Cur nanomicelles, and the solution was mixed uniformly 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.

[0044] Example 1: 0.007g of CMCS-FPBA was added to 10 mL of deionized water to obtain a 0.7% CMCS-FPBA solution, and 0.025g of β-TCP was added to 2.5 mL of the 0.7% CMCS-FPBA solution, and mixed uniformly to obtain solution A.

[0045] 0.01 g of PPS-HA nanomicelles was dissolved in 10 mL of PBS; 0.01 g of curcumin (Cur) was dissolved in 2 mL of an acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporation was performed for 3-5 min (40°C, 45 rpm), ultrasonic treatment was performed for 10 min, and a 0.45 um microporous filter membrane was used to prepare PPS-HA@Cur nanomicelles.

[0046] 0.1 g of ODex was added to 2.5 mL of the PPS-HA@Cur nanomicelles, and the solution was mixed uniformly to obtain solution B.

[0047] Solution A and solution B were mixed, and the pH was adjusted to 7-8 to finally form a microgel assembly.

[0048] Example 2: 0.007g of CMCS-FPBA was added to 10 mL of deionized water to obtain a 0.7% CMCS-FPBA solution, and 0.05g of β-TCP was added to 2.5 mL of the CMCS-FPBA solution, and mixed uniformly to obtain solution A.

[0049] 0.02 g of PPS-HA nanomicelles was dissolved in 10 mL of PBS; 0.02 g of Cur was dissolved in 2 mL of an acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporation was performed for 3-5 min (40°C, 45 rpm), ultrasonic treatment was performed for 10 min, and a 0.45 um microporous filter membrane was used to prepare PPS-HA@Cur nanomicelles.

[0050] 0.1 g of ODex was added to 2.5 mL of the PPS-HA@Cur nanomicelles, and the solution was mixed uniformly to obtain solution B.

[0051] Mix solution A with solution B, adjust pH to 7-8, finally form microgel assembly.

[0052] Example 3: 0.007g CMCS-FPBA was added into 10 mL deionized water to obtain a 0.7% CMCS-FPBA solution, and 0.025g β-TCP was added into 2.5 mL CMCS-FPBA solution to obtain solution A.

[0053] 0.01g of PPS-HA nanomicelles was dissolved in 10 mL PBS, and 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 evaporation was performed for 3-5 min (40°C, 45 rpm), ultrasonic treatment was performed for 10 min, and 0.45 um microporous filter membrane was used to prepare PPS-HA@Cur nanomicelles.

[0054] 0.2g of ODex was added into 2.5 mL PPS-HA@Cur nanomicelles, and the solution was mixed uniformly to obtain solution B.

[0055] Mix solution A with solution B, adjust pH to 7-8, finally form microgel assembly.

[0056] Example 4: 0.014g CMCS-FPBA was added into 10 mL deionized water to obtain a 1.4% CMCS-FPBA solution, and 0.025g β-TCP was added into 2.5 mL CMCS-FPBA solution to obtain solution A.

[0057] 0.01g of PPS-HA nanomicelles was dissolved in 10 mL PBS, and 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 evaporation was performed for 3-5 min (40°C, 45 rpm), ultrasonic treatment was performed for 10 min, and 0.45 um microporous filter membrane was used to prepare PPS-HA@Cur nanomicelles.

[0058] 0.1g of ODex was added into 2.5 mL PPS-HA@Cur nanomicelles, and the solution was mixed uniformly to obtain solution B.

[0059] Mix solution A with solution B, adjust pH to 7-8, finally form microgel assembly.

[0060] Example 5: 0.014 g of CMCS-FPBA was added to 10 mL of deionized water to obtain a 1.4% CMCS-FPBA solution, and 0.05 g of β-TCP was added to 2.5 mL of the CMCS-FPBA solution, which was mixed uniformly to obtain solution A.

[0061] 0.02 g of PPS-HA nanomicelles was dissolved in 10 mL of PBS, and 0.02 g of Cur was dissolved in 2 mL of an acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporation was performed for 3-5 min (40 °C, 45 rpm), ultrasonic treatment was performed for 10 min, and a PPS-HA@Cur nanomicelle was prepared by passing through a 0.45 um microporous filter membrane.

[0062] 0.2 g of ODex was added to 2.5 mL of the PPS-HA@Cur nanomicelle, and the solution was mixed uniformly to obtain solution B.

[0063] Solution A and solution B were mixed, and the pH was adjusted to 7-8 to finally form a microgel assembly.

[0064] Example 6: 0.014 g of CMCS-FPBA was added to 10 mL of deionized water to obtain a 1.4% CMCS-FPBA solution, and 0.05 g of β-TCP was added to 2.5 mL of the CMCS-FPBA solution, which was mixed uniformly to obtain solution A.

[0065] 0.02 g of PPS-HA nanomicelles was dissolved in 10 mL of PBS, and 0.02 g of Cur was dissolved in 2 mL of an acetone / ethanol solution (3:2); the organic phase liquid was added to the aqueous phase, rotary evaporation was performed for 3-5 min (40 °C, 45 rpm), ultrasonic treatment was performed for 10 min, and a PPS-HA@Cur nanomicelle was prepared by passing through a 0.45 um microporous filter membrane.

[0066] 0.2 g of ODex was added to 2.5 mL of the PPS-HA@Cur nanomicelle, and the solution was mixed uniformly to obtain solution B.

[0067] Solution A and solution B were mixed, and the pH was adjusted to 7-8 to finally form a microgel assembly.

[0068] Example 7: 0.02 g of CMCS-FPBA was added to 10 mL of deionized water to obtain a 2% CMCS-FPBA solution, and 0.05 g of β-TCP was added to 2.5 mL of the CMCS-FPBA solution, which was mixed uniformly to obtain solution A.

[0069] Take 0.02 g of PPS-HA nanomicelles dissolved in 10 mL of PBS; take 0.02 g of Cur dissolved in 2 mL of acetone / ethanol solution (3:2); add the organic phase liquid to the aqueous phase, rotary evaporation for 3-5 min (40 ℃, 45 rpm), ultrasonic for 10 min, pass through 0.45 um microporous filter membrane, and prepare PPS-HA@Cur nanomicelles.

[0070] Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA@Cur nanomicelles, mix the solution uniformly to obtain solution B.

[0071] Mix solution A with solution B, adjust the pH to 7-8, and finally form a microgel assembly.

[0072] Comparative Example 1: Take 0.014 g of CMCS-FPBA and add it to 10 mL of deionized water to obtain a 1.4% CMCS-FPBA solution, take 2 mg of β-TCP and add it to 2.5 mL of CMCS-FPBA solution, mix uniformly to obtain solution A.

[0073] Take 0.2 g of ODex and add it to solution A, adjust the pH to 7-8, and finally form a microgel assembly.

[0074] Comparative Example 2: Take 0.014 g of CMCS-FPBA and add it to 10 mL of deionized water to obtain a 1.4% CMCS-FPBA solution, take 2 mg of β-TCP and add it to 2.5 mL of CMCS-FPBA solution, mix uniformly to obtain solution A.

[0075] Take 0.02 g of PPS-HA and dissolve it in 10 mL of PBS to obtain PPS-HA nanomicelles. Take 0.2 g of ODex and add it to 2.5 mL of PPS-HA nanomicelles, mix the solution uniformly to obtain solution B.

[0076] Mix solution A with solution B, adjust the pH to 7-8, and finally form a microgel assembly.

[0077] Comparative Example 3: Take 0.014 g of CMCS-FPBA and add it to 10 mL of deionized water to obtain a 1.4% CMCS-FPBA solution, take 2 mg of β-TCP and add it to 2.5 mL of CMCS-FPBA solution, mix uniformly to obtain solution A.

[0078] Take 0.02 g of Cur and dissolve it in 2.5 mL of a mixed solution of DMSO and water (1:4), add 0.2 g of ODex, mix uniformly to obtain solution B.

[0079] Mix solution A with solution B, adjust pH to 7-8, finally form microgel assembly.

[0080] 1. Injectable image of hydrogel Figure 1 An injectable image of the hydrogel of Example 6 is shown.

[0081] 2. Self-healing image of hydrogel Figure 2 A self-healing image of the hydrogel of Example 6 is shown.

[0082] 3. Viscosity test Test method: inject the hydrogel onto the test sample table of the rotary rheometer, set the test temperature to 37 °C, the shear frequency to 1 Hz, and use a stainless steel lamella to test the viscosity of the liquid. The results are shown in Table 1.

[0083] Table 1. Viscosity test results

[0084] Results: As can be seen from Table 1, the viscosity of the hydrogel is affected by the ratio of CMCS-FPBA to ODex. When the content of CMCS-FPBA is 0.7% and the content of ODex is 4%, the viscosity of the hydrogel is 66.15 Pa / s, which ensures that the adhesion of the hydrogel to the bone tissue is sufficient to prevent the hydrogel from falling off or shifting, and ensures that it can continue to play a role in the repair process.

[0085] However, a hydrogel with too low viscosity may flow too quickly in the body, affecting its durability and drug release control in the body. A hydrogel with too high viscosity (such as Example 7) may clog the injection needle, making injection difficult. In Example 6, the hydrogel synthesized with a ratio of 0.7% CMCS-FPBA to 4% ODex was the most smooth to inject, and among them Figure 1 and Figure 2 are the schematic diagrams of the injectable image and the self-healing image of the hydrogel in Example 6. The other examples of hydrogel also present similar appearance.

[0086] 4. Degradation performance test Test method: first freeze-dry the prepared microgel assembly at -80 °C. Weigh and record the initial weight of the hydrogel after freeze-drying (the mass of the gelatin sponge remains the same), denoted as W0. Place the prepared freeze-dried hydrogel sample in a 24-well plate, add a fixed volume of collagenase solution with a pipette to completely immerse the hydrogel sample, and place it in a 37 °C constant temperature shaker. After 12 h, remove the sample, discard the excess degradation solution, freeze-dry, weigh, and record the weight, denoted as W t . The degradation rate is calculated according to the following formula:

[0087] Table 2. Degradation performance analysis

[0088] The results show that from Table 2, the degradation rate of the hydrogel of Example 6 of the present application in the collagenase degradation solution is 40.14±2.04% at 12h, which shows that the hydrogel of the present application has good stability and anti-degradation performance, and can better adapt to the complex oral environment.

[0089] The release amount of curcumin Test method: First, take different concentrations of curcumin (500, 250, 125, 62.5, 31.75 μg / mL) to establish a standard curve. Then prepare H2O2 solutions of different concentrations (0 mM, 0.25 mM, 0.5 mM), place 400 μL of microgels in Example 6 in 1 mL of H2O2 solution of a specific concentration (0.25 mM, 0.50 mM), and incubate in a constant temperature oscillation incubator at 37℃, 100 rpm. At different time points (1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 192 h), take samples, take 1 mL of culture solution of different H2O2 concentrations, and replace with an equal amount of fresh PBS. Centrifuge the sample at 4000 rpm for 5 min. The absorbance of curcumin in the PBS solution was measured at an excitation wavelength of 425 nm using ultraviolet spectrophotometry.

[0090] Calculation formula: Cumulative release rate (%) = (cumulative drug release amount / dosage) × 100% Figure 3 The cumulative release rate of curcumin is shown.

[0091] The results show that from Figure 3It can be seen that the release of Cur in PBS is much slower than that in H2O2, and the cumulative release amount is 49.4% at 192 h. However, the release rate of Cur is improved when incubated in 0.25 mM H2O2 solution, and the cumulative release amount is 69.3% at 192 h, showing high sensitivity to H2O2. The cumulative release amount is 88.9% at 192 h in 0.50 mM H2O2 solution, which significantly improves the release rate of Cur. This indicates that the concentration of H2O2 solution can accelerate the release rate of Cur. This may be due to the fact that PPS-HA nanomicelles undergo oxidation in the ROS environment, leading to the breakage of polymer chains or the destruction of cross-linked structures. This reaction promotes changes in the structure of micelles, resulting in faster release of curcumin from PPS-HA nanomicelles, which reflects its high adaptability to environmental changes. This ROS-responsive release mechanism enables the drug to be released at a specific time and place, and more importantly, this precise control of drug release rate by adjusting the concentration of ROS in the external environment can effectively improve the targeting and therapeutic effect of curcumin.

[0092] 6. Antioxidant capacity test Test method: The antioxidant properties of different examples and comparative examples were evaluated by the method of scavenging 1,1-diphenyl-2-pyridine hydrazine (DPPH) free radicals.

[0093] The mixture of different examples and comparative example samples and DPPH reagent was incubated in the dark for 30 minutes, and the remaining DPPH was analyzed by ultraviolet-visible spectroscopy. The formula for determining the DPPH scavenging rate is: D (%) = [[A 空白 - (A 测定 -A 对照 )] / A 空白 ]×100% Table 3. DPPH free radical scavenging rate (%)

[0094] Results: From Table 3, it can be seen that the PPS-HA-containing nanomicelles have free radical scavenging effect, which may be because the sulfur atoms in PPS-HA are easily oxidized by ROS to form sulfoxide, which is further oxidized to sulfone. Compared with small antioxidant molecules, PPS polymer contains multiple ROS reaction sites, which can scavenge more ROS. In addition, the free radical scavenging efficiency of PPS-HA nanomicelles containing Cur is higher than that of hydrogel containing Cur alone. This is because the phenolic hydroxyl groups in Cur are strong free radical terminators, which can provide electrons to free radicals, thereby reducing the free radicals to form stable molecules. This shows that the hydrogel containing both Cur and PPS-HA has higher free radical scavenging capacity, which indicates that Cur and PPS-HA have antioxidant synergistic effect.

[0095] 7. In vitro antibacterial test Test method: After recovery, P. gingivalis, E. coli and S. aureus were cultured to the logarithmic growth phase, centrifuged, and the collected bacteria were resuspended in physiological saline. The final concentration of the bacterial solution was 1 x 10 8 CFU / mL. The corresponding hydrogel samples were added to the 24-well plate. 500 μL of physiological saline was added to the control group. Then 100 μL of diluted bacterial suspension and 1 mL of sterile physiological saline were added to each well, and incubated at 37 ℃ for 24 hours. After gradient dilution, they were plated on BA medium or LB agar plates for counting.

[0096] Table 4. In vitro antibacterial rate (%)

[0097] Results: From Table 4, it can be seen that the addition of PPS-HA can enhance the antibacterial performance of Cur. Since PPS-HA improves the solubility of Cur, it helps curcumin to better exert its antibacterial effect. In addition, the slow release of curcumin in the PPS-HA composite helps to maintain long-term antibacterial activity. 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 shows that Cur and PPS-HA may have a synergistic effect when they exist simultaneously, enhancing the overall antibacterial activity.

[0098] 8. Anti-inflammatory evaluation: Test method: take the SD rats and weigh, 2% sodium pentobarbital intraperitoneal injection anesthesia, supine position the rats are fixed on the mouse board, pull the mandible as far as possible to expose the maxillary dentition. Surgical knife cut the maxillary first molar mesial gingival, gently turn up the gingival flap, cotton ball hemostasis, expose the bone surface, remove the first molar mesial root mesial alveolar bone with a diameter of 2mm ball drill, to the depth of the needle completely immersed to determine the hole depth. Then the diameter 2mm, depth of 1mm crack drill trimming hole shape defect, continuous water spray during operation to prevent bone necrosis. According to the grouping during operation, implant the corresponding material, suture the gingiva. Postoperative intramuscular penicillin to prevent infection. 10 days after operation, the alveolar bone tissue sample is washed to remove blood in pre-cooled PBS (0.02mol / L, pH 7.0-7.2), weighed and reserved; 1.0g tissue block is moved into the glass homogenizer, 5mL pre-cooled PBS is added, and the grinding process is carried out on ice; the obtained homogenate is repeatedly frozen and thawed 2 times, the prepared homogenate is centrifuged at 5000xg for 5 minutes, and the supernatant is reserved for detection by ELISA kit.

[0099] Figure 4 The anti-inflammatory effect of the hydrogel is shown.

[0100] The results show that: Figure 4 Compared with the physiological saline group and comparative example 3, the loaded example 6 hydrogel scaffold can effectively reduce the expression level of inflammatory factors TNF-α, IL-6 and IL-1β, and provide a stable microenvironment for alveolar bone tissue.

[0101] As can be seen from the above, the hydrogel of the embodiment of the present application uses CMCS-FPBA and ODex as matrix material, introduces Cur and PPS-HA, endows the hydrogel with antibacterial, antioxidant and immunoregulatory functions, and through optimization of the component ratio, the obtained hydrogel has good injectability, stability and anti-degradation, so that it can better play a role in repairing alveolar bone in the complex oral environment.

Claims

1. An injectable hydrogel for alveolar bone repair, characterized in that, It includes a first component and a second component. The first component includes 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan (CMCS-FPBA) and β-tricalcium phosphate. The second component includes oxidized dextran, PPS-HA nanomicelles, and curcumin.

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 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, FPBA, EDC, and NHS were dissolved in DMSO and stirred to activate the carboxyl groups to obtain an FPBA solution. Add the carboxymethyl chitosan solution to the FPBA solution, adjust the pH to 12, and stir overnight to react; After the reaction was complete, the solution was dialyzed; after dialysis, the supernatant was filtered and freeze-dried to obtain the 4-carboxy-3-fluorophenylboronic acid modified carboxymethyl chitosan CMCS-FPBA.

6. The injectable hydrogel according to any one of claims 1 to 4, characterized in that, The oxidized glucose is 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, react in the dark, add ethylene glycol to terminate the reaction, continue stirring, dialyze, and freeze dry to collect.

7. The injectable hydrogel according to any one of claims 1 to 4, characterized in that, 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 to dilute the mixture. The reaction solution 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.

8. A method for preparing an injectable hydrogel as described in any one of claims 1 to 7, 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; 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 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 glucose ODex to PPS-HA@Cur nanomicelles and mixing them evenly to obtain solution B; mixing solution A and solution B evenly and adjusting the pH to 7-8 to obtain the injectable hydrogel.

9. The use of the injectable hydrogel as described in any one of claims 1 to 7 in the preparation of antioxidant, anti-inflammatory and bone regeneration promoting drugs.

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

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