Enzyme-responsive composite hydrogels for periodontal tissues, their preparation methods and applications

Abstract

This invention discloses an enzyme-responsive composite hydrogel for periodontal tissues, its preparation method, and its applications, belonging to the field of biomedical materials technology. The composite hydrogel uses methacrylamide gelatin as a matrix, loaded with sodium ascorbate phosphate and calcium citrate; wherein calcium citrate accounts for 20-40% of the mass of methacrylamide gelatin, and the mass ratio of calcium citrate to sodium ascorbate phosphate is 1:0.8-1.5. This composite hydrogel can respond to enzyme signals in the periodontal microenvironment and synergistically release active ingredients to promote periodontal tissue regeneration, and can be used as a periodontal regeneration and repair material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an enzyme-responsive composite hydrogel for periodontal tissues, its preparation method, and its application. Background Technology

[0002] Periodontitis is a common chronic inflammatory disease that leads to irreversible destruction of periodontal supporting tissues, including alveolar bone. Currently, clinical repair of bone defects caused by periodontitis mainly relies on basic treatments (such as scaling and root planing) and surgical interventions (such as guided tissue regeneration and bone grafting). However, existing methods struggle to truly reconstruct the physiologically functional cementum-periodontal ligament-alveolar bone complex. The core challenge lies in the persistent vicious cycle of inflammation-bone destruction within the periodontal microenvironment. Plaque biofilm stimulation triggers an excessive immune response, leading to abnormally elevated levels of pro-inflammatory factors (such as TNF-α and IL-1β) and a large accumulation of reactive oxygen species (ROS), creating an oxidative stress microenvironment. M1 macrophage infiltration further releases matrix metalloproteinases (MMPs) and RANKL (receptor activator of nuclear factor κB), directly degrading the periodontal collagen matrix and activating osteoclasts, exacerbating alveolar bone resorption. At the same time, inflammatory factors and ROS jointly inhibit osteoblast differentiation, while calcium and phosphorus ion metabolism disorders hinder the mineralization and deposition of functional bone matrix, leading to a severe imbalance between bone formation and resorption.

[0003] Traditional single-treatment strategies are limited in effectiveness due to neglecting the systemic interference of the pathological microenvironment: simple anti-inflammatory treatments (such as local sustained-release antibiotics) can suppress inflammation in the short term, but cannot provide the osteogenic signals and mineralization conditions required for bone regeneration; simple implantation of mineralizing materials (such as calcium phosphate ceramics), while possessing osteoinductive potential, struggles to achieve functional regeneration within the inflammatory microenvironment. The fundamental reason lies in the continuous disruption of osteoblast activity by the inflammatory microenvironment, while mineralization defects cannot reverse immune imbalance. Therefore, overcoming regeneration bottlenecks and developing multifunctional scaffold materials that can respond to the pathological microenvironment and synergistically release active ingredients to promote periodontal tissue regeneration has become an urgent need in the field of periodontal regeneration. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an enzyme-responsive composite hydrogel for periodontal tissues, its preparation method, and its application. This composite hydrogel can respond to enzyme signals in the periodontitis microenvironment and synergistically release active ingredients to promote periodontal tissue regeneration, and can be used as a periodontal regeneration and repair material.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: an enzyme-responsive composite hydrogel for periodontal tissue is provided, wherein the composite hydrogel uses methacrylamide gelatin as a matrix and loads sodium ascorbate phosphate and calcium citrate; wherein, calcium citrate accounts for 20-40% of the mass of methacrylamide gelatin, and the mass ratio of calcium citrate to sodium ascorbate phosphate is 1:0.8-1.5.

[0006] The beneficial effects of this invention are as follows: The composite hydrogel of this invention uses methacrylamide gelatin (GelMA) as the matrix material and sodium ascorbate phosphate (SAP) and calcium citrate (CaC) as functional materials, forming an intelligent release system (SAP / CaC@GelMA) with dual-enzyme (acid phosphatase ACP / alkaline phosphatase ALP) responsive characteristics. This composite hydrogel can respond to enzymatic hydrolysis in the periodontal microenvironment, realizing the release of vitamin C (VC) and calcium citrate. 2+ / PO4 3- The synergistic release of these substances has multiple functions, including anti-oxidation, immunomodulation, and promoting bone mineralization.

[0007] GelMA exhibits excellent biocompatibility and cell adhesion, providing a scaffold for periodontal tissue regeneration. Furthermore, the three-dimensional network structure formed through photocrosslinking can serve as a carrier for sodium ascorbate phosphate and calcium citrate.

[0008] During the active phase of periodontitis, infiltrating inflammatory cells (such as M1 macrophages) release large amounts of acid phosphatase (ACP) and matrix metalloproteinases (MMPs). ACP is highly active in a slightly acidic inflammatory environment, creating a local acidic microenvironment that accelerates the hydrolysis of the GelMA backbone. The combined action of MMPs and the local acidic environment cleaves the molecular chains of GelMA, causing the hydrogel network structure to become loose or even locally disintegrate, thereby releasing the internally loaded active ingredients.

[0009] Sodium ascorbate phosphate (SAP), a phosphorylated derivative of vitamin C, is loaded onto a composite hydrogel. Upon release, it is hydrolyzed by acid phosphatase (ACP) and alkaline phosphatase (ALP), forming free vitamin C and phosphate ions (PO4). 3- Calcium citrate (CaC) dissociates and releases Ca in an acidic microenvironment. 2+ Vitamin C can remove excess reactive oxygen species from periodontal lesions, reduce oxidative stress damage to periodontal tissues, promote M2 macrophage polarization, and inhibit M1 pro-inflammatory phenotypes, thereby alleviating inflammation; it can also promote collagen synthesis. Because the mass ratio of SAP to CaC is 1:0.8-1.5, the released PO4... 3- and Ca 2+ It will deposit on collagen to form hydroxyapatite crystals, which directly promote alveolar bone regeneration.

[0010] In summary, the periodontal microenvironment contains inflammation (high ACP, acidity, oxidative stress) and bone defects (requiring ALP for mineralization). In environments with high ACP activity, the degradation rate of GelMA accelerates. Under acidic conditions and MMPs, GelMA releases the active components SAP and CaC. SAP is hydrolyzed by ACP or ALP, forming VC and PO4. 3- CaC releases Ca in an acidic environment 2+ Vitamin C plays an antioxidant and immunomodulatory role in inflamed areas, reducing inflammation and promoting collagen synthesis; PO4 3- and Ca 2+ Simultaneous release into the bone defect area can meet the needs of bone regeneration.

[0011] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, calcium citrate accounts for 30% of the mass of methacrylamide gelatin.

[0012] Furthermore, the mass ratio of calcium citrate to sodium ascorbate phosphate is 1:1.163.

[0013] The above-mentioned method for preparing enzyme-responsive composite hydrogels for periodontal tissues includes the following steps: To prepare a photoinitiator solution, methacrylamide gelatin was dissolved in the photoinitiator solution, then calcium citrate was added and mixed well. Sodium ascorbate phosphate was then added and mixed well. The mixture was then heated and stirred at 60-70℃ for 10-20 minutes, and finally irradiated with light to obtain a composite hydrogel.

[0014] Furthermore, the photoinitiator solution is prepared by the following method: dissolving the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in phosphate buffer solution (PBS solution), and stirring and heating at 40-50℃ for 10-20 min.

[0015] Further, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate was dissolved in PBS solution and heated at 45°C for 15 min to obtain the product.

[0016] Furthermore, the mass fraction of the photoinitiator solution is 0.2-0.3%, preferably 0.25%.

[0017] Furthermore, the mass fraction of methacrylated gelatin after dissolving in the photoinitiator solution is 5-10%, preferably 5%.

[0018] Further, after adding sodium ascorbate phosphate, mix well and heat at 65°C for 20 minutes with stirring.

[0019] Furthermore, the illumination conditions were 405nm light for 60s.

[0020] The above-mentioned enzyme-responsive composite hydrogels for periodontal tissues are used in the preparation of drugs for treating periodontitis or drugs or medical devices that promote periodontal tissue regeneration.

[0021] Furthermore, promoting periodontal tissue regeneration includes one or more of the following: inducing macrophages to polarize to the M2 type, anti-oxidation to reduce local inflammatory response, and promoting alveolar bone defect repair.

[0022] Furthermore, the medical device is an injectable bone filling material, a periodontal regeneration scaffold, or a drug sustained-release carrier.

[0023] The present invention has the following beneficial effects: (1) Functional synergy: SAP is decomposed by phosphatase to produce VC and PO4. 3- Vitamin C can scavenge reactive oxygen species (ROS), promote macrophage polarization towards the M2 anti-inflammatory type, and promote collagen synthesis; PO4 3- It can provide calcium and phosphorus sources required for bone formation together with CaC; the combination of SAP and CaC can achieve a synergistic effect of immune regulation and osteogenic mineralization.

[0024] (2) Intelligent release: Using the change of ACP / ALP level in the periodontal microenvironment as a trigger signal, SAP decomposition is triggered to realize VC and PO4. 3- On-demand, time-sequential release improves the precision of treatment.

[0025] (3) Performance enhancement: The introduction of SAP and CaC not only endows them with bioactivity, but also enhances the mechanical strength of the GelMA network as physical cross-linking points, making them more suitable for bone defect repair.

[0026] (4) Convenient operation: Based on the injectable and photocurable properties of GelMA, it can fit irregular defects and form them quickly in situ, making it highly clinically applicable. Attached Figure Description

[0027] Figure 1 SEM images of the GCS composite hydrogel and the control group hydrogel.

[0028] Figure 2 Results on the injectability and photocurability of the GCS composite hydrogel precursor solution.

[0029] Figure 3 The absorption spectra of the extracts of GCS composite hydrogel and control group hydrogel are shown.

[0030] Figure 4Image showing F-actin staining of GCS composite hydrogel and PDLSCs cells co-cultured, scale bar 200 μm; Con represents the control.

[0031] Figure 5 The stress-strain curves are shown for the GCS composite hydrogel and the control group hydrogel.

[0032] Figure 6 The results are from the FTIR measurements of the GCS composite hydrogel.

[0033] Figure 7 The macroscopic performance of DPPH free radical scavenging ability of GCS composite hydrogel and control group hydrogel extracts.

[0034] Figure 8 The graph shows the quantitative data on the DPPH free radical scavenging capacity of the extracts of GCS composite hydrogel and control group hydrogels.

[0035] Figure 9 The results of Chem 3D simulations show the interaction between SAP and CaC in GCS composite hydrogels.

[0036] Figure 10 The ALP / ARS staining results for each group of hydrogel extracts are shown; CON is the control.

[0037] Figure 11 The results show the migration of PDLSCs in the hydrogel extracts of each group.

[0038] Figure 12 The results of quantitative statistical analysis of the migration rate of PDLSCs in the hydrogel extracts of each group were presented.

[0039] Figure 13 The images show the fluorescent staining of the DCFH-DA probe after co-culturing the hydrogel extracts with cells in each group.

[0040] Figure 14 The statistical results of ROS clearance under natural breathing conditions are as follows: Figure 13 The quantitative results.

[0041] Figure 15 The effect of different concentrations of H2O2 on PDLSC S Cell damage.

[0042] Figure 16 The image shows the fluorescent staining of the DCFH-DA probe after co-culturing cells with GCS composite hydrogel extract after H2O2 treatment; CON is the control group of well plates treated with H2O2 but without extract, and NC is the conventionally cultured cell group without H2O2 treatment.

[0043] Figure 17The results show the fluorescence intensity of cells after co-culturing with GCS composite hydrogel extract after H2O2 treatment; CON is the control group of well plates treated with H2O2 but without extract, and NC is the conventionally cultured cell group without H2O2 treatment. Detailed Implementation

[0044] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0045] Example 1: A method for preparing an enzyme-responsive composite hydrogel for periodontal tissues includes the following steps: (1) Accurately weigh each component: 250.0 mg of methacrylamide gelatin (GelMA), 107.1 mg of calcium citrate (CaC), 124.6 mg of sodium ascorbate phosphate (SAP), and 50 mg of photoinitiator (LAP).

[0046] (2) Preparation of photoinitiator solution (LAP solution): Dissolve 50 mg LAP in 20 mL of PBS solution with pH=7.4, and then stir and heat in a 45℃ magnetic water bath for 15 min.

[0047] (3) Dissolution and mixing: Add GelMA, CaC and SAP to the container in sequence, then add 4.5 mL of LAP solution filtered through a 0.22 μm filter. Rinse the container wall with the remaining 0.5 mL of LAP solution. Then place the container in a 65℃ water bath and stir magnetically at 500 rpm for 20 min until all components are completely mixed to obtain a uniformly dispersed suspension, which is the GCS composite hydrogel precursor solution.

[0048] (4) Photocrosslinking curing: Dispense the suspension into 24-well plates at a rate of 500 μL / well. Immediately place the 24-well plates under a 405 nm UV lamp, keeping the lamp source 10 cm away from the liquid surface, and irradiate evenly for 60 s. After irradiation, a stable hydrogel can be seen rapidly formed in the solution.

[0049] The enzyme-responsive composite hydrogel for periodontal tissue prepared above is denoted as CaC / SAP@GelMA composite hydrogel (hereinafter referred to as GCS composite hydrogel).

[0050] Control group setup: Pure GelMA hydrogel (G hydrogel), CaC@GelMA hydrogel (GC hydrogel), SAP@GelMA hydrogel (GS hydrogel). The control groups differed from Example 1 only in the added functional component; all other components remained unchanged. Details are as follows: Pure GelMA hydrogel (G hydrogel): Unlike Example 1, the gel composition contains only GelMA and does not contain CaC and SAP. The rest of the process is the same as in Example 1.

[0051] CaC@GelMA hydrogel (GC hydrogel): The difference from Example 1 is that the gel composition contains GelMA and CaC, but does not contain SAP. The rest of the process is the same as in Example 1.

[0052] SAP@GelMA hydrogel (GS hydrogel): The difference from Example 1 is that the gel composition contains GelMA and SAP, but does not contain CaC. The rest of the process is the same as in Example 1.

[0053] The following tests were performed on the above hydrogel: I. Morphological Observation The GCS composite hydrogel obtained in Example 1 of this invention and the hydrogel obtained in the control group were first subjected to liquid nitrogen brittle fracture (the purpose of which is to obtain a smooth fracture surface while maintaining the morphology and structure for subsequent observation), then freeze-dried, and finally their morphology and structure were observed under a scanning electron microscope. The results are as follows. Figure 1 As shown.

[0054] Depend on Figure 1 It can be seen that the GCS composite hydrogel, G hydrogel, GC hydrogel, and GS hydrogel have uniform pore size and distribution, good porosity, and are suitable for cell growth and ion exchange. This indicates that the functional components introduced into the hydrogel do not affect the basic structure of the hydrogel.

[0055] II. Injectability and Photocurability Testing The injectability test of the GCS composite hydrogel precursor solution obtained in step (3) of Example 1 of the present invention was carried out by manual extrusion experiment (qualitative observation). Specifically, under room temperature conditions, the GCS composite hydrogel precursor solution was loaded into a 1mL syringe, pre-cured under 405nm light for 15s, and then injected to observe the extrusion continuity and morphological stability of the hydrogel.

[0056] The specific process of the photocurability test (macroscopic performance) is as follows: the GCS composite hydrogel precursor solution is loaded into a transparent vial, the vial is tilted, and its state before and after 405nm photocuring is observed.

[0057] The results of the injectability and photocurability tests of the GCS composite hydrogel precursor solution are as follows: Figure 2 As shown. By Figure 2 It can be seen that the GCS composite hydrogel precursor solution can become solid after 60s of 405nm light irradiation and can become fluid-like substance after 15s of pre-curing, with good injectability and photocurability.

[0058] III. Two-enzyme response assay The specific process for performing a dual-enzyme response assay on the GCS composite hydrogel prepared in Example 1 of this invention and the control group is as follows: The solid GCS composite hydrogel obtained by solidifying 1 mL of GCS composite hydrogel precursor solution was mixed with 500 μL of alkaline phosphatase (ALP, 50 U / mL) and treated at 37℃ for 1 h. The resulting liquid was recorded as GCS-ALP. Separately, 1 mL of the solid GCS composite hydrogel obtained by solidifying the precursor solution was mixed with 500 μL of acid phosphatase (ACP, 50 mg / mL) and treated at 37℃ for 1 h. The resulting liquid was recorded as GCS-ACP. G hydrogel, GS hydrogel, and GCS composite hydrogel were treated only with 500 μL of phosphate buffer at 37℃ for 1 h. Finally, the absorption spectra were measured at 260 nm using an ultra-micro spectrophotometer. The results are as follows: Figure 3 As shown.

[0059] Depend on Figure 3 It can be seen that the absorbance of the GCS composite hydrogel treated with ALP or ACP is significantly higher than that of the control group hydrogel, indicating that the active ingredient SAP in the GCS composite hydrogel is decomposed by enzymes to release vitamin C, indicating that the GCS hydrogel has ALP / ACP dual enzyme response characteristics.

[0060] IV. Biocompatibility Testing The GCS composite hydrogel prepared in Example 1 of this invention was co-cultured with periodontal ligament stem cells (PDLSCs). The specific procedure was as follows: the GCS composite hydrogel precursor solution was injected into a 24-well plate and cured under 405 nm light for 60 s. Then, the plate was rinsed three times with α-MEM complete culture medium (containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin), 5 min each time. Finally, the culture was diluted with 1×10⁻⁶... 5 Periodontal ligament stem cells were implanted into each well, with medium changes every two days, and the culture medium was always α-MEM complete medium. The control group (G hydrogel, GC hydrogel, and GS hydrogel) was also implanted using the same method. F-actin staining was performed on day 4, and the results are as follows... Figure 4 As shown.

[0061] Depend on Figure 4 It can be seen that PDLSCs can adhere and spread well on GCS composite hydrogel, indicating that GCS composite hydrogel has good biocompatibility.

[0062] V. Mechanical Property Testing The stress-strain curves of the GCS composite hydrogel prepared in Example 1 of this invention and the control group hydrogel were measured. The specific procedure was as follows: 2 mL of each group of hydrogel solution was taken and prepared into a substrate with a bottom area of ​​approximately 1.767 cm². 2A cylinder with a height of approximately 1.13 cm was subjected to quantitative strain testing using an EFL-MT5600 hydrogel microforce tester. Specifically, stress changes were observed and the mechanical strength of each hydrogel group was analyzed at 50% strain. The results are as follows: Figure 5 As shown.

[0063] Depend on Figure 5 It can be seen that the elastic modulus of GC hydrogel and GS hydrogel is increased compared with that of G hydrogel, while that of GCS composite hydrogel is increased compared with that of GC hydrogel and GS hydrogel. This indicates that the introduction of CaC and SAP can significantly increase the mechanical properties of GelMA.

[0064] VI. Infrared Spectroscopy Measurement The GCS composite hydrogel, pure GelMA hydrogel, raw material CaC, and SAP prepared in Example 1 of this invention were subjected to Fourier transform infrared (FTIR) spectroscopy. In addition, another control group was set up: the GCS composite hydrogel was treated with ALP (the treatment method was the same as in the previous enzyme response assay, then the gel block was freeze-dried to prepare a powder, denoted as GCS / ALP), and then FTIR was performed. The results are as follows. Figure 6 As shown.

[0065] Depend on Figure 6 It can be seen that the GCS composite hydrogel treated with ALP has a viscosity of 1000-1100 cm⁻¹. -1 The presence of characteristic peaks, which correspond to the position of the characteristic absorption peaks of phosphate ions, further verifies the enzymatic effect of GCS hydrogel.

[0066] VII. Determination of DPPH free radical scavenging ability Prepare reaction solutions for G, GC, GS, GCS, GCS-ALP, and GCS-ACP groups according to the procedure described above for the dual-enzyme response assay. Mix each reaction solution with the working solution in the DPPH free radical scavenging kit for antioxidant capacity testing (96-well plate, 20 μL sample solution + 80 μL buffer + 100 μL DPPH working solution per well). Then, place the plate at 25℃ and incubate in the dark for 3 min. Finally, measure the absorbance at 517 nm using a microplate reader to obtain specific values. Quantify the scavenging rate using the formula: Scavenging rate = [(DPPH working solution - X) / DPPH working solution] × 100% (where X is the specific value of the other groups, e.g., DPPH standard solution value is 0) (DPPH standard solution is a calibration sample with a scavenging capacity of 10% in the kit). The results are as follows: Figure 7 and 8 As shown.

[0067] Depend on Figure 7 and 8It was found that all groups loaded with SAP exhibited significant DPPH radical scavenging ability, indicating that SAP loading can endow the hydrogel with antioxidant activity. However, the antioxidant activity of the GCS group was not as good as that of the GS group, suggesting that the presence of CaC and the release of SAP may have a mutual restraint. But when ALP or ACP was introduced, the enzymatic decomposition of SAP endowed the GCS hydrogel with good antioxidant activity, indirectly confirming the dual-enzyme response dynamic characteristics of the GCS composite hydrogel.

[0068] VIII. Determination of the interaction between SAP and CaC The interaction between SAP and CaC in the GCS composite hydrogel prepared in Example 1 of this invention was simulated using Chem 3D software (the molecular structures of SAP and CaC were plotted in Chem 3D software, and the potential forces were calculated using the software's built-in MM2 force field simulation function). The results are shown in […]. Figure 9 .

[0069] Depend on Figure 9 It is known that SAP and CaC may form a relatively stable structure through ionic and hydrogen bond interactions. (Combined) Figure 7 and 8 The results show that the GCS group is not as good as the GS group, which further illustrates that SAP and CaC in the GCS hydrogel system are not just a simple mixture, but may form a sustained-release effect through mutual restraint.

[0070] IX. Measurement of bone-promoting properties Extracts were prepared from the GCS composite hydrogel obtained in Example 1 of this invention and a control group to test the hydrogel's properties. The specific process for preparing the extracts is as follows: GCS composite hydrogel, G hydrogel, GC hydrogel and GS hydrogel were placed in complete culture medium and then placed in a cell culture incubator and incubated at 37°C in the dark for 72 h. The extracts were collected and filtered through a 0.22 μm filter and stored at 4°C.

[0071] The complete culture medium mentioned above includes: α-MEM medium and the following components at final concentrations: 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0072] The amount of hydrogel added was calculated with reference to the extraction ratio of 0.1 g / mL specified in GB / T14233.2-2025 Test Methods for Medical Infusion, Transfusion and Injection Equipment Part 2: Biological Test Methods.

[0073] The extracts of the GCS composite hydrogel obtained in Example 1 of this invention and the extracts of the control group hydrogel were prepared according to the above process. These were then used to prepare corresponding osteogenic induction media (taking GCS osteogenic induction liquid as an example, the component concentrations in the GCS osteogenic induction liquid were: dexamethasone 0.1 μM, L-ascorbic acid 50 μg / mL, β-glycerophosphate sodium 10 mM, 10% fetal bovine serum, 1% penicillin-streptomycin solution, and the remaining liquid was GCS extract). Periodontal ligament stem cells (PDLSCs) were cultured with the medium changed every two days, using a half-medium change method. ALP / ARS staining was performed on days 7 (Day 7) and 14 (Day 14), and the results are as follows. Figure 10 As shown.

[0074] Control group (CON): Compared with the experimental group, the CON group had the complete culture medium replaced with the corresponding extract, while the concentrations of the other components remained unchanged, namely dexamethasone 0.1 μM, L-ascorbic acid 50 μg / mL, β-glycerophosphate sodium 10 mM, 10% fetal bovine serum, and 1% penicillin-streptomycin solution.

[0075] Depend on Figure 10 As can be seen from ALP staining, the GCS composite hydrogel exhibits stronger alkaline phosphatase activity. ARS staining also reveals more calcified nodule deposition. These results indicate that the GCS composite hydrogel possesses stronger osteogenic properties.

[0076] 10. Cell migration assay Following the procedures described above for osteogenic performance testing, extracts of the GCS composite hydrogel prepared in Example 1 of this invention and extracts of the control group hydrogel were prepared. Then, well-grown P3 generation PDLSCs (5 × 10⁶ cells) were seeded in 6-well plates. 5 (Cells / well) When the cell density reaches approximately 85%, use a sterile pipette tip to make an "I" shaped scratch along the diameter of the well plate. Then rinse three times with PBS to remove dead cells, 3 minutes each time. Add 2 mL of the corresponding extraction solution to each well according to the group. The CON group receives α-MEM complete medium (10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin). Incubate at 37℃ in a 5% CO2 incubator. At 0H (0 hours), 12H (12 hours), and 24H (24 hours), photographs of the same area are taken under an inverted microscope and recorded. ImageJ software is used to quantify the scratch area. The results are shown below. Figure 11 and 12 As shown.

[0077] Depend on Figure 11 and 12 It can be seen that the extract of GCS composite hydrogel has a stronger ability to promote the migration of PDLSCs.

[0078] XI. Determination of Reactive Oxygen Scrap Capacity To simulate the generation of reactive oxygen species (ROS) by cells under natural physiological conditions through metabolism such as the mitochondrial respiratory chain, the ROS scavenging ability of the composite hydrogel of this invention under natural cellular respiration was evaluated. The specific process is as follows: Following the procedures described above for osteogenic performance testing, extracts of the GCS composite hydrogel prepared in Example 1 of this invention and extracts of the control group hydrogel were prepared. P3 generation PDLSCs in good growth condition were taken, and the cell density was adjusted to 1×10⁻⁶. 5 One sample per well was seeded into a 12-well plate. 1 mL of the corresponding extraction solution was added to each well according to the group. For the CON group, α-MEM complete medium (10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin) was added instead of the extraction solution. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, all liquid was aspirated, and the plates were rinsed twice with PBS. 0.5 mL of the prepared DCFH-DA working solution was added to each well, and the plates were incubated at 37°C in the dark for 30 minutes. After the reaction, the plates were washed twice with PBS buffer. The fluorescence of DCFH-DA was observed and photographed under an inverted fluorescence microscope. The fluorescence intensity was quantitatively analyzed using ImageJ software. The results are shown below. Figure 13 and 14 As shown.

[0079] Depend on Figure 13 and 14 It can be seen that GCS composite hydrogel has good antioxidant properties under the natural respiration state of cells.

[0080] XII. Oxidative Stress Measurement To further simulate the oxidative stress generated by cells under inflammatory conditions, hydrogen peroxide was used to induce cellular oxidative stress. The specific process is as follows: (1) H2O2-induced oxidative stress damage to PDLSC S Establishment of cell models PDLSCs in the logarithmic growth phase S Cells at 1×10 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The old culture medium was then discarded, and 100 μL of complete culture medium containing 0, 50, 100, 150, 200, 250, 300, 350, and 400 μmol / L H2O2, respectively, were added and incubated at 37°C with 5% CO2 for 24 h. Cell viability was measured using the CCK8 assay after incubation. Results are shown below. Figure 15 As shown.

[0081] When establishing an oxidative stress model, if cell viability is too low, it will cause irreversible cell damage; if cell viability is too high, it cannot induce significant oxidative stress damage. Therefore, a damage range of approximately 50% cell viability is chosen. According to... Figure 15 As a result, 150 μmol / mL H2O2 was selected as the treatment concentration for the oxidative stress model in this experiment.

[0082] (2) GCS composite hydrogel on oxidative stress-damaged PDLSCs S Cell protection Following the procedures described above for osteogenic performance testing, extracts of the GCS composite hydrogel prepared in Example 1 of this invention and extracts of the control group hydrogel were prepared. P3 generation PDLSCs in good growth condition were taken, and the cell density was adjusted to 1×10⁻⁶. 5 Each well was seeded with one sample of the corresponding extract. The samples were then incubated at 37°C for 24 hours in a 5% CO2 incubator. The old culture medium was then discarded, and both the CON group and the experimental groups were incubated with 150 μmol / mL H2O2 complete medium and incubated at 37°C for 24 hours in a 5% CO2 incubator. The old medium was discarded, and the samples were washed three times with PBS. Then, 1 mL of the corresponding extraction solution was added to each well according to the group. For the CON group, α-MEM complete medium (10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin) was used instead of the extraction solution, and the samples were incubated at 37°C for 24 hours in a 5% CO2 incubator. After incubation, all liquid was aspirated, and the plate was rinsed twice with PBS. The prepared DCFH-DA working solution was added to each well of a 12-well plate (0.5 mL). The plate was incubated at 37°C in the dark for 30 min. After the reaction, the plate was washed twice with PBS buffer. The fluorescence of DCFH-DA was observed and photographed under an inverted fluorescence microscope. The fluorescence intensity was quantitatively analyzed using ImageJ software. The results are shown below. Figure 16 and 17 As shown.

[0083] Depend on Figure 16 and 17 It is evident that GCS composite hydrogels exhibit excellent reactive oxygen species scavenging capabilities under cellular oxidative stress.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An enzyme-responsive composite hydrogel for periodontal tissues, characterized in that, The composite hydrogel uses methacrylamide gelatin as a matrix and is loaded with sodium ascorbate phosphate and calcium citrate; wherein, calcium citrate accounts for 20-40% of the mass of methacrylamide gelatin, and the mass ratio of calcium citrate to sodium ascorbate phosphate is 1:0.8-1.

5.

2. The enzyme-responsive composite hydrogel for periodontal tissues according to claim 1, characterized in that, Calcium citrate accounts for 30% of the mass of methacrylamide gelatin.

3. The enzyme-responsive composite hydrogel for periodontal tissues according to claim 1, characterized in that, The mass ratio of calcium citrate to sodium ascorbate phosphate is 1:1.

163.

4. The method for preparing the enzyme-responsive composite hydrogel for periodontal tissue according to any one of claims 1 to 3, characterized in that, Includes the following steps: To prepare a photoinitiator solution, methacrylamide gelatin was dissolved in the photoinitiator solution, then calcium citrate was added and mixed well. Sodium ascorbate phosphate was then added and mixed well. The mixture was then heated and stirred at 60-70℃ for 10-20 minutes, and finally irradiated with light to obtain a composite hydrogel.

5. The preparation method according to claim 4, characterized in that, The photoinitiator solution was prepared by dissolving lithium phenyl-2,4,6-trimethylbenzoylphosphonate in a phosphate buffer solution and stirring and heating at 40-50°C for 10-20 minutes.

6. The preparation method according to claim 4 or 5, characterized in that, The mass fraction of the photoinitiator solution is 0.2-0.3%.

7. The preparation method according to claim 4, characterized in that, The mass fraction of methacrylated gelatin after dissolving in a photoinitiator solution is 5-10%.

8. The preparation method according to claim 4, characterized in that, After adding sodium ascorbate phosphate, mix well and heat at 65°C for 20 minutes with stirring.

9. The preparation method according to claim 4, characterized in that, The illumination conditions were 405nm light for 60 seconds.

10. The use of the enzyme-responsive composite hydrogel for periodontal tissue according to any one of claims 1 to 3 in the preparation of a medicament for treating periodontitis.

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