Injectable hydrogel as well as preparation method and application thereof

Abstract

The invention relates to injectable hydrogel and a preparation method and application thereof.The injectable hydrogel is composed of a rigid network and a flexible network, the flexible network is prepared from poly N-isopropylacrylamide (PNIPAM), and the rigid network is prepared from a metal polyphenol coordination compound; wherein the metal polyphenol coordination compound is formed by coordination of demethylated lignin and divalent metal ions. The injectable hydrogel provided by the invention adopts the combination of the formamide, the ammonia water and the aqueous solution of hydrogen peroxide, so that the injectable hydrogel is relatively low in toxicity, can be easily and cleanly removed in manners of reduced pressure distillation, freeze drying and the like, and is relatively good in biological safety.

Description

Technical Field

[0001] The present invention relates to an injectable hydrogel, in particular to a hydrogel that can be used for treating periodontitis. Background Art

[0002] Periodontitis is a common and frequently occurring oral disease. Uncontrolled periodontitis is the leading cause of tooth loss in adults, affecting over 19% of the population and severely impacting oral and systemic health. Periodontitis is a chronic, infectious, and progressive disease of the periodontium. In response to bacterial and metabolic products, the periodontium produces excessive amounts of reactive oxygen species (ROS), which cause local oxidative stress in the periodontium, exacerbating inflammation and tissue destruction. Peri-implantitis is a common complication after implant restorations, characterized by inflammatory destruction of the soft tissue surrounding the implant and progressive resorption of the supporting bone. With a global incidence of up to 22%, it poses a serious threat to the long-term stability of the implant and oral function. Its pathogenesis is closely related to microbial infection mediated by bacterial biofilms, an imbalance in the host immune-inflammatory response, and an oxidative stress microenvironment. Conventional clinical treatment for periodontitis and peri-implantitis involves mechanical cleaning supplemented with systemic or topical antibiotics. However, rapid recolonization of the bacterial biofilm after mechanical debridement can lead to recurrence of periodontitis in susceptible patients, while excessive antibiotic use can cause a variety of systemic adverse reactions. Although the existing common commercially available local treatment formulations for periodontitis and peri-implantitis, minocycline hydrochloride ointment, have certain antibacterial and anti-inflammatory effects, in the unique environment of the oral cavity, the continuous secretion of saliva, rinsing, and swallowing can easily lead to the premature consumption of the locally delivered periodontal treatment formulation, making it difficult to achieve a sustained and effective therapeutic concentration at the site of inflammation. Although a formulation with too high a concentration can achieve a transient therapeutic effect, it is prone to potential toxic side effects, and traditional treatment methods mainly remove the cause of the disease, and lack support for anti-oxidative stress, anti-inflammation, and promoting bone tissue repair. Therefore, there is an urgent need to explore newer and more effective treatment strategies that can enable the locally delivered formulations to adhere stably to the periodontal and peri-implant tissues for a long time, while being able to efficiently and continuously remove excess free radicals produced by the periodontal tissues and perform sustained and effective sterilization, thereby correcting the inflammatory microenvironment and improving the outcome of periodontitis.

[0003] Lignin is a naturally derived polyphenolic substance with antioxidant and macrophage polarization regulating functions. Demethylation of lignin can significantly improve the antioxidant properties of lignin. At the same time, its abundant phenolic hydroxyl groups can form metal polyphenol networks (MPNs) with a variety of metal ions. MPNs are pH-responsive and can respond to the acidic microenvironment of periodontal and peri-implantitis to achieve responsive sustained release of metal ions and demethylated lignin. However, traditional demethylation methods are inefficient and have poor biosafety, such as the use of toxic reagents such as thiols, sodium sulfide, halogen acids, acidic molten salts, halides, and aluminum chloride. Therefore, the application of the above demethylation methods in the biomedical field is greatly limited.

[0004] Poly (N-isopropylacrylamide) (PNIPAM) is a temperature-sensitive polymer material. Its unique temperature responsiveness enables it to have good fluidity at room temperature, and solidify at physiological temperature (37°C) to adapt to the complex periodontal pocket environment. It has broad application prospects in the field of periodontitis, but the function of simple hydrogel is limited, and its antioxidant, anti-inflammatory, osteogenesis impaired, and antibacterial functions are insufficient. Summary of the Invention

[0005] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides an injectable hydrogel. The hydrogel in the present invention adopts a combination of formamide, ammonia water, and an aqueous solution of hydrogen peroxide. It has low toxicity and can be easily removed by reduced pressure distillation, freeze drying, etc., and has better biosafety.

[0006] According to a first aspect of the present invention, the present invention provides an injectable hydrogel, characterized in that the injectable hydrogel is composed of a rigid network and a flexible network, wherein the flexible network is prepared from poly (N-isopropylacrylamide) (PNIPAM), and the rigid network is prepared from a metal polyphenol coordination compound; wherein the metal polyphenol coordination compound is formed by coordination of demethylated lignin and divalent metal ions, and the preparation method of the demethylated lignin is as follows: S11: mixing lignin and formamide and heating them to obtain component 1; S12: adding ammonia water and hydrogen peroxide to component 1 in sequence, stirring under predetermined stirring conditions to obtain component 2; S13: performing reduced pressure distillation on component 2 to remove unreacted formamide to obtain component 3; S14: freeze-drying component 3 to obtain demethylated lignin.

[0007] In a preferred embodiment, in step S1, the volume ratio of lignin mass to formamide is (0.05 g-0.2 g):1 mL.

[0008] In a preferred embodiment, the heating temperature in step S1 is 40-65°C.

[0009] In a preferred embodiment, the predetermined stirring condition is stirring at 300-800 rpm for 1-4 hours.

[0010] In a preferred embodiment, the molecular weight of the lignin is 8-15 kDa.

[0011] In a preferred embodiment, the divalent metal ion may be a calcium ion, a magnesium ion or a copper ion.

[0012] According to a second aspect of the present invention, the present invention provides a method for preparing an injectable hydrogel, characterized in that the preparation method comprises the following steps: S21: Dissolve PNIPAM in DMF and heat to obtain component 4, wherein the mass ratio of PNIPAM to DMF is 20 mg:1 mL; S22: The metal polyphenol coordination compound was added to component 4 and dried under vacuum to obtain component 5; S23: Add water to component 5 to swell the component to obtain a hydrogel.

[0013] In a preferred embodiment, the preparation method of the metal polyphenol coordination compound is as follows: dissolve demethylated lignin in water, adjust the pH to 9.0-9.5, raise the temperature to 55-65°C, slowly and uniformly add divalent metal ion solution and mix well to obtain the metal polyphenol coordination compound.

[0014] In a preferred embodiment, the preparation method of the metal demethylated lignin is as follows: S11: mixing lignin and formamide and heating them to obtain component 1; S12: adding ammonia water and hydrogen peroxide to component 1 in sequence, stirring under predetermined stirring conditions to obtain component 2; S13: performing reduced pressure distillation on component 3 to remove unreacted formamide; S14: Freeze-drying to obtain demethylated lignin.

[0015] According to a third aspect of the present invention, the present invention provides a use of an injectable hydrogel in preparing a product for treating periodontitis.

[0016] Beneficial effects of the present invention: 1. The lignin demethylation method provided by the present invention can demethylate lignin safely, non-toxicly and mildly, thereby improving the antioxidant capacity of lignin.

[0017] 2. The injectable hydrogel provided by the present invention can be advantageously applied to periodontal pockets of periodontitis to inhibit bacterial growth, effectively remove excess ROS caused by periodontitis, promote macrophage M2 polarization, and promote alveolar bone regeneration.

[0018] 3. This injectable thermosensitive hydrogel, formed by self-assembling a rigid network formed by demethylated lignin coordinated with various metal ions and a flexible PNIPAM network, can uniformly and slowly release demethylated lignin and metal ions in a simulated periodontal microenvironment, providing long-term relief from oxidative stress, immune regulation, and bone formation, thereby treating alveolar bone loss caused by periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a Fourier transform infrared spectrum of the demethylated lignin prepared in Example 1; Figure 2 is the H NMR spectrum of the demethylated lignin prepared in Example 1; Figure 3 This is the result of testing phenolic hydroxyl content by Folin-phenol method; Figure 4 The CCK-8 method is used to detect the demethylated lignin (DL), iodocyclohexane demethylated lignin (DL-ICH), sodium sulfite demethylated lignin Figure 1 shows the toxicity results on gingival fibroblasts; Figure 5 The release results of the main components of the injectable hydrogel at different pH values are shown in Figure 2, where (a) is the release result of demethylated lignin (DL) and (b) is the release result of calcium ions. Figure 6 This is the result of detecting reactive oxygen species in cells (RAW264.7 cells and MC3T3-E1 cells) using DCFH-DA probe; Figure 7 This is the result of macrophage iNOS and CD206 immunofluorescence staining to characterize macrophage polarization; Figure 8 This is the result of ELISA test of macrophage anti-inflammatory factors and pro-inflammatory factors; Figure 9 This is the result of testing L and DL antioxidant activity using DPPH, ABTS, and PTIO methods; Figure 10 This is the result of the antibacterial ability of the injectable hydrogel, with the control group on the left and the hydrogel group on the right; Figure 11 is an optical image of the injectable hydrogel injectability; Figure 12The figures are performance test diagrams of injectable hydrogels at different temperatures, wherein (a) is the thermosensitive performance test result diagram of injectable hydrogels at 22°C, and (b) is the thermosensitive performance test result diagram of injectable hydrogels at 37°C; Figure 13 This is a scanning electron micrograph of the injectable hydrogel. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0021] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0022] Example 1: Preparation of demethylated lignin 1. Mix 1 g of lignin with 8 mL of formamide and heat to 40°C for 6 hours. Stir at 300 rpm for 30 minutes to fully swell to obtain component 1. 2. Slowly add 16 mL of 25% aqueous ammonia to component 1, raise the temperature to 70°C, maintain the pH at 10-11, and stir at 300 rpm for 10 minutes. Then, add 300 μL of 3% hydrogen peroxide dropwise at a rate of 2 mL / min. Under nitrogen, stir at 300 rpm for 1 hour to obtain component 2. 3. Distill at 75°C, -0.09 MPa vacuum to remove unreacted formamide to obtain component 3; 4. Freeze-dry component 3 at -20°C to obtain demethylated lignin.

[0023] The demethylated lignin prepared in Example 1 was subjected to Fourier transform infrared spectroscopy (FTIR) spectroscopy. The spectroscopy was performed using a Spectrum 400 (PerkinElmer, USA) instrument at a wavelength range of 500 to 4000 nm. Within 0.5 The FTIR spectra of lignin and demethylated lignin were obtained by 32 scan accumulations and resolution. Figure 1 The results showed that the methoxyl group was at 1120 The vibration is weakened, phenolic hydroxyl 1218 The vibration becomes stronger, indicating that the lignin after demethylation treatment contains more phenolic hydroxyl groups and thus has better antioxidant capacity.

[0024] The demethylated lignin prepared in Example 1 was subjected to NMR testing using a Bruker 400 MHz NMR instrument. Spectrum acquisition, test temperature is 25 ° C, solvent is .

[0025] Test results see Figure 2 , combined with Figure 2 It can be seen that in In the spectrum, it can be seen that the characteristic methoxy proton peak at 3.3 ppm in the DL sample is significantly reduced, indicating that the methoxy content in the demethylated lignin is lower.

[0026] The total phenol content in lignin and DL was determined by the Folin-Ciocalteu colorimetric method. The specific method was as follows: 1 mL of acetic acid-zinc acetate solution (10:1, v / v) was added to 1 mg of lignin and 1 mg of demethylated lignin prepared in Example 1, and mixed thoroughly. Then, 5 mL of Folin-phenol reagent was added, mixed and allowed to stand for 5 minutes, and then 4 mL of saturated The solution was mixed and allowed to stand at room temperature in the dark for 1 h. The absorbance was measured at 760 nm using an ultraviolet spectrophotometer, and the total phenolic hydroxyl content in the sample was calculated using a standard curve of guaiacol of known concentration.

[0027] Test results see Figure 3 ,from Figure 3 It can be seen that the demethylation treatment increased the phenolic hydroxyl content of lignin from 3.3 mmol / g to 4.7 mmol / g. The above experiments illustrate the successful implementation of demethylation.

[0028] Comparative Example 1 5g of lignin was dissolved in 10 mL of double distilled water and stirred for 10 minutes. Then, 0.75g of The mixed solution was reacted at 100°C with vigorous stirring for 1 hour. Double-distilled water was added to terminate the reaction. After cooling to room temperature, dilute hydrochloric acid was added dropwise to adjust the pH of the solution to approximately 2.0. DL was purified by dialysis and then dried under vacuum at 55°C to obtain DL powder.

[0029] Comparative Example 2 0.5 g of lignin was dissolved in 12.5 mL of N,N-dimethylformamide (DMF), and 3.5 mL of ICH reagent was added to the solution. The mixture was heated to 130°C in a sealed container for 12 hours. After the reaction, the reaction mixture was cooled, extracted with n-hexane, and then added dropwise to 125 mL of 2 M HCl solution with vigorous stirring for 3 hours. Finally, the resulting precipitate was washed with deionized water until neutral and dried in vacuo at 50°C to obtain DL-ICH.

[0030] Example 2: Preparation of Metal Polyphenol Coordination Compound A 1. Dissolve 1g of demethylated lignin in 500mL of water (in a round-bottom flask), adjust the pH to 9.5, and heat to 60℃. 2. Add 0.25g The solution was dissolved in 20 mL of deionized water to prepare a calcium ion solution, and the calcium ion solution was evenly added to the round-bottom flask at a rate of 1 mL / min and stirred at 800 rpm for 10 minutes to obtain the metal polyphenol coordination compound A.

[0031] The demethylated lignin in this example is the demethylated lignin prepared in Example 1.

[0032] Example 3: Preparation of Metal Polyphenol Coordination Compound B 1. Dissolve 1g of demethylated lignin in 500mL of water, adjust the pH to 9.5, and heat to 60℃. 2. Dissolve 0.2 g of MgCl2 in 20 mL of deionized water to prepare a magnesium ion solution. Add the magnesium ion solution evenly to the round-bottom flask at a rate of 1 mL / min and stir at 800 rpm for 10 minutes.

[0033] The demethylated lignin in this example is the demethylated lignin prepared in Example 1.

[0034] Example 4: Synthesis of Metal Polyphenol Coordination Compound C 1. Dissolve 1g of demethylated lignin in 500mL of water (in a round-bottom flask), adjust the pH to 9.5, and heat to 60℃. 2. Add 0.1g The copper ion solution was dissolved in 20 mL of deionized water to prepare a copper ion solution, and the copper ion solution was evenly added to the round-bottom flask at a rate of 1 mL / min and stirred at 800 rpm for 10 minutes to obtain the metal polyphenol coordination compound C.

[0035] The demethylated lignin in this example is the demethylated lignin prepared in Example 1.

[0036] Example 5: Preparation of injectable hydrogel The injectable hydrogel is composed of a rigid network and a flexible network, wherein the flexible network is prepared from poly N-isopropylacrylamide (PNIPAM) and the rigid network is prepared from a metal polyphenol coordination compound. The preparation method of the injectable hydrogel is as follows: 1. Dissolve 2 g of PNIPAM in 100 mL of DMF (100% purity) and heat to 60°C to obtain component 4. 2. Add 2 mL of the metal polyphenol coordination compound A solution obtained in Example 2 dropwise to component 4 at a constant rate of 1 mL / min. Stir at 600 rpm for 2 hours and then vacuum dry at 60°C for 24 hours to obtain component 5. 3. Add 100 mL of 4°C water to component 5 to swell it. Swell it overnight at a speed of 10 mm / s to obtain an injectable hydrogel.

[0037] Example 6 Cytotoxicity Test of Different Demethylated Lignins The fourth generation of periodontal ligament stem cells PDLSCs were used Cells / well were seeded in a 96-well plate and pre-cultured for 24 hours before being added to three groups of culture media (DMEM medium supplemented with 1% penicillin-streptomycin and 5% fetal bovine serum), wherein the three groups of culture media contained the demethylated lignin prepared in Example 1, Comparative Example 1, and Comparative Example 2, respectively, and the final concentration of the demethylated lignin in the three groups of culture media was 0.1 mg / mL; 3 replicates were set for each group, and CCK-8 solution was added after incubation for 48 hours in the dark for 2 hours. The absorbance at 450 nm was measured by a microplate reader, and the cell survival rate was calculated.

[0038] The test results can be found in Figure 4 ,from Figure 4 It can be seen that the DL group showed no obvious toxicity, and the cell survival rate was maintained above 96%. The survival rate of the group was only 80% at the same concentration, while the survival rate of DL-ICH was only 63%. This shows that the demethylated lignin synthesized by the demethylation method adopted in this application has better biocompatibility than the traditional demethylation method.

[0039] Example 7 Hydrogel pH responsiveness test 1 mL of the injectable hydrogel sample prepared in Example 5 was completely immersed in 1 L of PBS buffer (pH 7.4 / pH 6.3), and placed in a constant temperature shaker (horizontal reciprocating, amplitude 2 cm) at 37°C and 100 rpm for continuous shaking and incubation; 3 mL of the extract was aspirated using a pipette at a preset time point, filtered through a 0.22 μm filter membrane, and immediately supplemented with an equal amount of fresh PBS at the same temperature to maintain a constant volume of the system; a two-component standard curve was established using an ultraviolet spectrophotometer: calcium ion detection was performed using a calcium ion detection kit (Biyuntian S1063S), and a series of 0.05-2 mg / L standard solutions of demethylated lignin were prepared (at intervals of 0.1 mg / L). Three parallel samples were set for each concentration point, and the absorbance was measured at the characteristic absorption wavelength (320 nm for demethylated lignin). The linear regression equation was used. Calculate the actual concentration and perform background subtraction and interference peak correction simultaneously.

[0040] Test results see Figure 5 ,from Figure 5 It can be seen from (a) and (b) that the hydrogel slowly and evenly releases demethylated lignin and calcium ions within 7 days, and the release rate is significantly increased in a weakly acidic environment (pH = 6.3), indicating that the hydrogel has significant pH responsiveness.

[0041] Example 8 Reactive oxygen species (ROS) scavenging test MC3T3-E1 preosteoblasts were mixed with RAW 264.7 macrophages to The cells / well density was inoculated into 96-well black transparent bottom culture plates, and DMEM (containing 10% FBS) was used to culture at 37°C and 5% The cells were pre-cultured under the following conditions for 24 hours to 80% confluence; 1 μg / mL LPS, 1 μg / mL LPS+0.1 mg / mL DL, and 1 μg / mL LPS+10 mg / mL DL-Ca / PNIPAM were added to the experimental groups, respectively, and 6 replicate wells (including 3 biological replicates and 2 technical replicates) were set up in each group; after 6 hours of treatment, the culture medium was aspirated and discarded, and serum-free culture medium containing 10 μM DCFH-DA probe was added. The cells were incubated at 37°C in the dark for 30 minutes, and the culture plate was gently shaken every 10 minutes to ensure uniform contact of the probe; after removing the probe solution, 200 μL / well of pre-cooled PBS (4°C, pH 7.4) was slowly injected along the wall of the well, allowed to stand for 1 minute, and then aspirated and discarded. The washing was repeated 3 times to completely remove the extracellular probe; the cells were immediately incubated at the excitation wavelength Emission wavelength Fluorescence images were collected at different wavelengths.

[0042] The test results can be found in Figure 6 ,from Figure 6As can be seen, the fluorescence intensity of the LPS-treated group was significantly higher than that of the PBS-treated group, indicating that LPS induced the production of ROS. The fluorescence intensities of the LPS+DL and LPS+DL-Ca / PNIPAM-treated groups were both lower than those of the LPS-treated group, indicating that these two treatments can effectively eliminate ROS.

[0043] Example 9 RAW 264.7 cells were cultured with The cells were seeded in 96-well black transparent bottom culture plates and incubated in DMEM medium containing 10% FBS at 37°C. After culturing to 60% confluence, the experimental groups were treated with 1 μg / mL LPS, 1 μg / mL LPS + 0.1 mg / mL DL, and 1 μg / mL LPS + 10 mg / mL DL-Ca / PNIPAM for 24 hours. After discarding the culture medium, the cells were gently washed three times with pre-chilled PBS (2 minutes each time), fixed with 4% paraformaldehyde (pH 7.4, containing 0.1 M phosphate buffer) at room temperature for 15 minutes, and permeabilized with 0.1% Triton X-100 (dissolved in PBS containing 1% BSA) for 10 minutes. Nonspecific sites were blocked with 5% homologous serum (species-matched to the secondary antibody) at room temperature for 1 hour. Subsequently, anti-iNOS antibodies (1:200 dilution in blocking buffer) and anti-CD206 antibodies (1:100 dilution in blocking buffer) were added dropwise and incubated at 4°C in a humidified chamber for 16 hours. The cells were then warmed to 37°C for 1 hour the next day to enhance antigen-antibody binding. After removing the primary antibody, TBST buffer (containing 0.05% The slides were washed three times with Tween-20 (5 minutes each time) with shaking. Secondary antibodies (1:500 dilution) labeled with Alexa Fluor 488 anti-rabbit IgG (corresponding to iNOS detection) and Alexa Fluor 594 anti-mouse IgG (corresponding to CD206 detection) were added, respectively. After incubation at room temperature in the dark for 1 hour, the cell nuclei were counterstained with DAPI (1 μg / mL) for 5 minutes. Finally, the slides were covered with anti-fluorescence quenching mounting medium, and three-channel fluorescence signals (iNOS excitation / emission wavelengths 490 / 525 nm, CD206 555 / 580 nm, DAPI 358 / 461 nm) were collected under a laser confocal microscope with a 20× objective lens. Five fields of view were randomly selected from each well for Z-stack scanning (layer thickness 0.5 μm).

[0044] The test results can be found in Figure 7 ,from Figure 7It can be seen that in the LPS group, the expression of iNOS in the cell nucleus increased significantly, while the expression of CD206 decreased, indicating that LPS induced an inflammatory response in macrophages. Compared with the LPS group, the expression of inducible nitric oxide synthase (iNOS) decreased in the LPS+DL and LPS+DL-Ca / PNIPAM groups, while the expression of CD206 increased slightly, indicating that DL can alleviate the LPS-induced inflammatory response and promote the polarization of M2 macrophages.

[0045] Example 10 RAW 264.7 cells The wells were inoculated into 96-well plates and treated with 1 μg / mL LPS, 1 μg / mL LPS+0.1 mg / mL DL-Ca, and 1 μg / mL LPS+10 mg / mL DL-Ca / PNIPAM for 48 hours. The supernatant was collected and centrifuged to remove debris. The dilutions were mixed at a ratio of 1:4 and the concentrations of inflammatory factors were detected using TNF-α / IL-10 / TGF-β / iNOS ELISA kits according to the standard procedure: add 100 μL of sample → incubate at 37°C for 90 minutes → wash the plate 5 times → add 100 μL of biotinylated antibody → react at 37°C for 60 minutes → wash the plate and add HRP-labeled streptavidin → develop with TMB for 15 minutes → measure the OD value at 450 / 570 nm after termination.

[0046] The test results can be found in Figure 8 ,from Figure 8 It can be seen that LPS stimulation significantly increased the expression of pro-inflammatory factors: inducible nitric oxide synthase (iNOS) reached 132 pg / mL and tumor necrosis factor-α (TNF-α) reached 1086 pg / mL, while DL and DL-Ca / PNIPAM treatment reduced the two to 60-70 pg / mL and 420-450 pg / mL, respectively, while increasing the anti-inflammatory factor TGF-β to 300 pg / mL and IL-10 to 28-31 pg / mL. The data showed that DL-Ca / PNIPAM significantly enhanced the M2 polarization characteristics while effectively inhibiting M1 macrophage markers.

[0047] Example 11 The ability of lignin (purchased from Sigma-Aldrich, CAS No.: 8068-05-1) at different concentrations and the demethylated lignin prepared in Example 1 to scavenge DPPH, ABTS, and PTIO free radicals was detected using an ultraviolet spectrophotometer.

[0048] The test results can be found in Figure 9 ,from Figure 9 It can be seen that the addition of DL at the same concentration makes the color of the free radical lighter than that of L, indicating that DL exhibits significantly better free radical scavenging ability than L.

[0049] Example 12 Fusobacterium nucleatum was inoculated into BHI liquid medium and cultured at 37°C and 200 rpm with shaking until the logarithmic growth phase (OD600≈0.6). The cells were collected by centrifugation (4000 rpm, 10 min) and washed twice with sterile PBS. The concentration of the bacterial suspension was adjusted to ; Take 100 μL of bacterial solution and mix it with 100 mg of injectable hydrogel sample (injectable hydrogel prepared in Example 5) or sterile PBS (control group) in a 1.5 mL centrifuge tube, incubate at 37°C for 2 hours, then take 50 μL of the mixture and evenly spread it on a BHI plate. The plate was inverted and incubated in a constant temperature incubator at 37°C for 24 hours, and then counted by the four-quadrant colony counting method.

[0050] The test results can be found in Figure 10 ,from Figure 10 It can be seen that the colony in the hydrogel group was significantly reduced compared with the control group, indicating that the injectable hydrogel of the present invention has antibacterial ability.

[0051] Example 13 The injectable hydrogel prepared in Example 5 was injected onto the surface of an A4 paper at room temperature using a 1 mL syringe.

[0052] The test results can be found in Figure 11 ,from Figure 11 It can be seen that the hydrogel has good injectability and molding ability to be suitable for periodontal microenvironment application.

[0053] Example 14 The thermosensitive ability of the injectable hydrogel prepared in Example 5 was tested using the vial inversion method. The vial was tilted or inverted at 22° C. and 37° C., and optical photographs were taken.

[0054] The test results can be found in Figure 12 ,from Figure 12 As can be seen from (a) and (b), the hydrogel is fluid at room temperature (22°C) and solidifies and has certain adhesion at 37°C, which is beneficial for application in the complex periodontal microenvironment.

[0055] Example 15 The injectable hydrogel sample prepared in Example 5 was freeze-dried at -40°C for 24 hours to remove moisture and maintain its structural integrity. After freeze-drying, the sample was sputtered with a 3 nm gold layer using an ion sputtering instrument at a current of 15 mA and an argon atmosphere of 0.1 mbar. The sample was immediately transferred to the sample stage of a scanning electron microscope. A high-resolution secondary electron probe was used to capture multi-scale morphological images at 500× to 50,000× using backscattered electron imaging (BSE) mode at an acceleration voltage of 5 kV, a working distance of 8 mm, and a beam spot diameter of 3 nm.

[0056] The test results can be found in Figure 13 ,from Figure 13 It can be seen that the hydrogel is composed of a loose porous structure, which is beneficial to the sustained release of demethylated lignin and metal ions. At the same time, the high specific surface area enhances the adaptability of the hydrogel to the irregular periodontal environment.

[0057] In summary, the above are only some preferred specific embodiments of the present invention. Any corresponding changes and modifications made by those skilled in the art based on the concept of the present invention and the prior art shall fall within the scope of protection of the appended claims of the present invention.

Claims

1. An injectable hydrogel, characterized in that The injectable hydrogel is composed of a rigid network and a flexible network, wherein the flexible network is prepared from poly (N-isopropylacrylamide) and the rigid network is prepared from a metal polyphenol coordination compound; wherein the metal polyphenol coordination compound is formed by coordination of demethylated lignin and divalent metal ions, and the preparation method of the demethylated lignin is as follows: S11: mixing lignin and formamide and heating them to obtain component 1; S12: adding ammonia water and hydrogen peroxide to component 1 in sequence, stirring under predetermined stirring conditions to obtain component 2; S13: performing reduced pressure distillation on component 2 to remove unreacted formamide to obtain component 3; S14: freeze-drying component 3 to obtain demethylated lignin.

2. The injectable hydrogel according to claim 1, wherein in step S11, the volume ratio of lignin to formamide is 0.05 g-0.2 g:1 mL.

3. The injectable hydrogel according to claim 1, characterized in that The heating temperature in step S11 is 40-65°C.

4. The injectable hydrogel according to claim 1, wherein The predetermined stirring condition is stirring at 300-800 rpm for 1-4 hours.

5. The injectable hydrogel according to claim 1, characterized in that The molecular weight of the lignin in step S11 is 8-15 kDa.

6. The injectable hydrogel according to claim 1, characterized in that The divalent metal ions may be calcium ions, magnesium ions or copper ions.

7. The method for preparing an injectable hydrogel according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: S21: dissolving poly (N-isopropylacrylamide) in DMF and heating to obtain component 4, wherein the mass ratio of poly (N-isopropylacrylamide) to DMF is 20 mg:1 mL; S22: The metal polyphenol coordination compound was added to component 4 and dried under vacuum to obtain component 5; S23: Add water to component 5 to swell, thereby obtaining a hydrogel.

8. The method for preparing an injectable hydrogel according to claim 7, wherein: The preparation method of the metal polyphenol coordination compound is as follows: The demethylated lignin is dissolved in water, the pH value is adjusted to 9.0-9.5, the temperature is raised to 55-65° C., and a divalent metal ion solution is slowly and uniformly added dropwise and mixed to obtain a metal polyphenol coordination compound.

9. The method for preparing an injectable hydrogel according to claim 8, wherein: The preparation method of the demethylated lignin is as follows: S11: mixing lignin and formamide and heating them to obtain component 1; S12: adding ammonia water and hydrogen peroxide to component 1 in sequence, stirring under predetermined stirring conditions to obtain component 2; S13: performing reduced pressure distillation on component 2 to remove unreacted formamide; S14: freeze-drying to obtain demethylated lignin.

10. Use of the injectable hydrogel according to any one of claims 1 to 6 in preparing a product for treating periodontitis.

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