A new gel repair material, preparation method and application

Abstract

The application discloses a novel gel repair material, a preparation method and application, relates to the field of tissue repair gel, and aims to solve the problem of single function of the existing water gel, and adopts the technical scheme that DSPE-PEG modified sodium bicarbonate nanoparticles, PVP modified PB NPs and GelMA gel material are made into a composite hydrogel, and the concentrations of the three are controlled. The porous structure of GelMA is stable, PB NPs and sodium bicarbonate are slowly released, so as to control the release kinetics of the two, make the slow release of PB NPs continuously remove ROS and generate oxygen, the continuous release of sodium bicarbonate maintains the pH stability, realizes the time sequence synchronization of "ROS removal-oxygen supply-pH buffering", and further controls the concentrations of PB NPs and sodium bicarbonate, ensures that the oxygen release is in the platform period, can release oxygen at a stable rate, the pH is stable at 7.2-7.4, ensures that the function intensity of PB NPs and sodium bicarbonate is matched, and avoids local microenvironment imbalance, and breaks through the limitation of single function in the prior art.

Description

Technical Field

[0001] This invention relates to the field of tissue repair gel technology, specifically to a novel gel repair material, its preparation method, and its application. Background Technology

[0002] The immune microenvironment at bone defect sites is often disrupted by persistent inflammation induced by implants or bacterial invasion. Simultaneously, due to ruptured blood vessels and interrupted blood supply, the defect area often experiences persistent hypoxia, triggering a series of pathophysiological responses: hypoxia not only inhibits osteoblast differentiation and metabolic activity but also induces mitochondrial dysfunction, leading to excessive accumulation of reactive oxygen species (ROS). High levels of ROS promote osteoblast apoptosis and downregulate osteogenic gene expression, while simultaneously activating osteoclast-mediated bone resorption. The released acidic metabolites further exacerbate local tissue acidification, ultimately forming a vicious cycle of "hypoxia-ROS-acidification," severely hindering bone regeneration. These interconnected factors constitute a self-reinforcing negative cycle, significantly impairing osteogenic function and limiting the effectiveness of conventional treatment strategies. Therefore, developing bioactive materials capable of synergistically regulating the microenvironment of bone defect areas is of significant strategic importance for advancing the functional repair of bone defects.

[0003] Among numerous nanomaterials, Prussian blue nanoparticles (PB NPs) exhibit excellent physicochemical stability and biosafety. On one hand, they can directly capture and neutralize various ROS (such as oxygenates) through a unique electron transfer mechanism. , (etc.) effectively reduce the adverse effects of oxidative stress on the osteogenic process; on the other hand, PB NPs also have catalase-like activity, which can specifically catalyze the decomposition of hydrogen peroxide accumulated in bone defect areas into water and oxygen, thereby improving the hypoxic state locally while clearing ROS, thus providing a new solution for the simultaneous regulation of the dual microenvironmental barriers of "oxidation-hypoxia".

[0004] Another major obstacle in the bone repair microenvironment is local acidosis. Sodium bicarbonate ( Sodium bicarbonate exhibits unique advantages in regulating pH in bone defect areas. As a core component of the human endogenous buffering system, its mechanism of action is highly consistent with that of the bicarbonate buffer pair in the blood. It can precisely neutralize local hydrogen ions through bicarbonate ions, not only efficiently removing acidic substances, but also allowing the carbon dioxide produced by the reaction to be naturally excreted through the body's metabolic pathways, thus avoiding the risk of bubble accumulation. In addition, the biocompatibility of sodium bicarbonate has been verified through long-term clinical practice (such as its use in correcting metabolic acidosis), with no risk of foreign body reactions, and can provide a long-term, stable physiological acid-base microenvironment for cell growth, differentiation, and bone matrix mineralization.

[0005] Methacrylamide gelatin (GelMA) hydrogel has become an ideal material for constructing bone tissue engineering carriers due to its good biocompatibility, tunable degradation, tissue adhesion, and photocrosslinking processing capabilities. Its biomimetic microenvironment has been proven to effectively promote osteogenic processes in vivo.

[0006] Currently, most strategies for regulating the microenvironment of bone defects focus on single aspects such as scavenging reactive oxygen species (ROS), delivering oxygen, or regulating pH. However, directly integrating existing PB NPs with sodium bicarbonate to create multifunctional hydrogels presents numerous challenges, including issues with multi-component compatibility and the timing of functional synergy. Specifically, the enzyme-like activity of PB NPs may be affected by the alkaline environment of sodium bicarbonate; rapid release of sodium bicarbonate may disrupt the cross-linking structure of GelMA, leading to a decrease in the material's mechanical properties. Furthermore, excessively rapid oxygen release from PB NPs could result in excessively high local oxygen concentrations, while excessively rapid buffering by sodium bicarbonate could trigger a sudden pH spike. A mismatch in the release rates of the two components could also cause a rebound of a particular pathological factor. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the existing defects and provide a novel gel repair material, preparation method and application, which can effectively solve the problems in the background art.

[0008] To achieve the above objectives, this invention discloses a method for preparing a gel repair material, the technical solution of which includes the following steps: Step 1: Preparation of sodium bicarbonate nanoparticles. A chloroform solution for synthesizing sodium bicarbonate nanoparticles via microemulsion method; Step 2, modification of sodium bicarbonate nanoparticles, A chloroform solution of DSPE-PEG was added to a chloroform solution of sodium bicarbonate nanoparticles synthesized in step 1. The mixture was stirred and reacted at room temperature and then purified to obtain DSPE-PEG modified sodium bicarbonate nanoparticles. Step 3: Prepare PVP (polyvinylpyrrolidone) modified PB NPs; Step 4: Prepare composite gel repair material. The DSPE-PEG modified sodium bicarbonate nanoparticles obtained in step 2, the PVP-modified PB NPs obtained in step 3, and 10% GelMA gel material were mixed to prepare a composite gel repair material. In the composite gel repair material, the concentration of PB NPs caused the oxygen release rate to reach a plateau, and the concentration of DSPE-PEG modified sodium bicarbonate nanoparticles was able to stabilize the pH of the composite gel repair material at 7.2-7.4.

[0009] Sodium bicarbonate nanoparticles (approximately 200 nm in diameter) were synthesized using a microemulsion method and then surface-modified with DSPE-PEG to enhance their dispersibility in the GelMA matrix and prevent aggregation. A 10% concentration of GelMA gel material was used as the matrix, and the three-dimensional network formed after photocrosslinking could not only encapsulate the two types of nanoparticles but also maintain sufficient mechanical strength (Young's modulus meets the support requirements for skull defects) without affecting the sustained release of the active ingredients.

[0010] The controllable degradation and porous structure of GelMA were used to regulate the release kinetics of PB NPs and sodium bicarbonate: PB NPs were slowly released to continuously scavenge ROS and generate oxygen, while sodium bicarbonate continuously released bicarbonate to maintain pH stability, achieving a temporal synchronization of "ROS scavenging - oxygen supply - pH buffering"; the concentration of PB NPs was controlled at 50 μg / mL to allow oxygen release to reach a plateau, and the concentration of sodium bicarbonate was matched to maintain a stable pH of 7.2-7.4, ensuring that the functional strength of the two was matched and avoiding local microenvironmental imbalance.

[0011] As a preferred technical solution of the present invention, in step 1, sodium oleate, anhydrous ethanol, oleylamine and n-hexane are mixed and ultrasonically dispersed, then ammonium bicarbonate aqueous solution is added, stirred at room temperature to carry out the reaction, centrifuged after the reaction is completed, the precipitate obtained by separation is washed with anhydrous ethanol, and then dispersed in chloroform to obtain a chloroform solution of sodium bicarbonate nanoparticles.

[0012] As a preferred embodiment of the present invention, in step 2, the mixed solution is continuously stirred and reacted at room temperature for 12 h, and then vacuum dried at 40°C to obtain the product.

[0013] As a preferred embodiment of the present invention, in step 3, polyvinylpyrrolidone and potassium ferricyanide are dissolved in an ethanol solution containing HCl to obtain a reactive precursor solution. The reactive precursor solution is placed in a water bath at 80°C and reacted for 3 hours before being cooled to room temperature. The resulting dark blue reaction mixture is washed with an ultrafiltration tube and freeze-dried to obtain PVP-modified PB NPs.

[0014] The preparation method of this PB NPs can obtain particles with uniform size (particle size of about 70 nm) and stable physicochemical properties, ensuring that they still maintain catalase-like activity and ROS scavenging ability in the presence of sodium bicarbonate.

[0015] As a preferred embodiment of the present invention, in step 4, GelMA, DSPE-PEG modified sodium bicarbonate nanoparticles, and PVP-modified PB NPs are added to a PBS solution containing a photoinitiator, and a composite gel is prepared by ultraviolet light irradiation.

[0016] The present invention also discloses a gel repair material prepared by the above method, which can be used for biological tissue repair operations including bone tissue repair, and can construct an anti-inflammatory repair microenvironment during the repair process, while taking into account angiogenesis and osteogenic processes.

[0017] Compared with existing technologies, the advantages of this invention are as follows: This invention synthesizes sodium bicarbonate nanoparticles using a microemulsion method and modifies them with DSPE-PEG to enhance the dispersibility of sodium bicarbonate in a GelMA matrix, preventing aggregation and excessively high local alkalinity. PB NPs are modified with PVP to form uniformly sized and chemically stable particles, ensuring they retain catalase-like activity and ROS scavenging ability in the presence of sodium bicarbonate. Using 10% GelMA as the matrix, the three-dimensional network formed after photocrosslinking can encapsulate both types of nanoparticles while maintaining sufficient mechanical strength and not affecting the sustained release of the active ingredient. These measures together solve the compatibility issues between PB NPs, sodium bicarbonate, and GelMA.

[0018] Furthermore, the porous structure of GelMA allows for the stable and slow release of PB NPs and sodium bicarbonate, thereby regulating their release kinetics. This enables PB NPs to slowly release and continuously scavenge ROS and generate oxygen, while sodium bicarbonate continuously releases bicarbonate ions to maintain pH stability, achieving temporal synchronization of "ROS scavenging - oxygen supply - pH buffering." Moreover, by controlling the concentrations of PB NPs and sodium bicarbonate, oxygen release is ensured to be at a plateau, allowing for a stable oxygen release rate and maintaining a stable pH of 7.2-7.4. This ensures a functional match between PB NPs and sodium bicarbonate, preventing local microenvironmental imbalance.

[0019] Furthermore, PB NPs and the pH buffer sodium bicarbonate were integrated into the GelMA hydrogel system to form... Nanocomposite hydrogels. This composite system achieves comprehensive regulation of the bone defect microenvironment through the synergistic action of multiple mechanisms: PB NPs, as highly efficient ROS scavengers, can convert hydrogen peroxide into oxygen through their catalase-like activity, alleviating oxidative stress while improving local hypoxia and inhibiting osteoclast activation; sodium bicarbonate provides a continuous and stable pH buffering capacity, effectively neutralizing acidic metabolites, combating local acidosis, and stabilizing the pH at the optimal osteogenic range of 7.2-7.4, avoiding the inhibition of osteoblast differentiation by the acidic environment, and eliminating the short-term buffering defects of sodium bicarbonate when used alone; while GelMA hydrogel, as a biocompatible extracellular matrix-like scaffold, not only provides three-dimensional support for cell adhesion and osteogenic differentiation, but also constructs an ideal carrier for oxygen transport and microenvironment regulation.

[0020] Furthermore, PB NPs in this novel gel repair material also have the ability to regulate the immune microenvironment, promoting macrophage polarization towards the M2 phenotype and further creating an anti-inflammatory and regenerative local environment. The oxygen generated by PB NPs, together with the stable pH environment of sodium bicarbonate, enables HUVEC cells to form a more complete tubular network, with significantly higher branch node numbers and tube lengths than the single PB NPs / hydrogel group. At the same time, it enhances the migration ability of osteogenic progenitor cells (MC3T3-E1), achieving "angiogenesis-osteoogenesis" coupling and solving the problem that single functional materials cannot simultaneously address both vascularization and osteoogenesis.

[0021] Furthermore, in a rat model of critical skull defects, the novel gel repair material prepared in this invention showed a bone volume fraction (BV / TV) three times higher than the single-component hydrogel group at 8 weeks, and a significantly higher bone mineral density (BMD), forming a cortical structure more closely resembling natural bone. Histological examination revealed that the novel gel repair material group exhibited significant new bone formation at 4 weeks, and formed a thickened soft tissue layer and osteoid structure at 8 weeks, with no inflammation or damage to major organs, demonstrating its efficient osteogenic properties and biocompatibility in vivo. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the construction and bone regeneration promotion of the novel gel repair material of this invention; Figure 2 This is a characterization diagram of the novel gel repair material of the present invention; Figure 2 a) Fourier transform infrared spectra of sodium bicarbonate, DSPE-PEG and surface-modified sodium bicarbonate nanomaterials; Figure 2 b) is a particle size distribution of sodium bicarbonate measured by dynamic light scattering; Figure 2 c) is a particle size distribution diagram of PB nanoparticles measured by dynamic light scattering; Figure 2 d) is an image of the novel gel repair material prepared by photopolymerization; Figure 2 e) A comparison of Young's modulus of hydrogels with different GelMA concentrations (10%, 15%, 20%, 25%); Figure 2 f) is for GelMA, PB / GelMA and Comparison of Young's modulus of hydrogels; Figure 2 g) is for GelMA, PB / GelMA and Scanning electron microscope image of the cross-section of the composite hydrogel; Figure 2 h) is GelMA after 24 hours. PB / GelMA and Comparison of swelling ratios of composite hydrogels; Figure 2 i) GelMA within 14 days, PB / GelMA and Comparison of degradation rates of composite hydrogels; Figure 3 This diagram illustrates the characteristics of PB NPs and the pH-regulating effect of sodium bicarbonate nanoparticles in this invention. Figure 3 a) Schematic diagram of DPPH clearing mechanism; Figure 3 b) UV-Vis absorption spectra of DPPH solution at different time points; Figure 3 c) is a graph showing the DPPH free radical scavenging efficiency over time; Figure 3 d) is Schematic diagram of free radical scavenging mechanism; Figure 3 e) for different time points UV-Vis absorption spectrum of the solution; Figure 3 f) represents the time-varying value. Free radical scavenging efficiency graph; Figure 3 g) is a comparison chart of oxygen produced by hydrogen peroxide decomposition at different PB concentrations as measured by a dissolved oxygen meter; Figure 3 h) is a graph showing the relationship between different sodium bicarbonate concentrations and the pH conditions they can create; Figure 3 i) is the cumulative sodium ion release curve of sodium bicarbonate nanoparticles in Tris-HCl buffer at pH=6; Figure 4 This is a diagram showing the cell compatibility test results of the novel gel repair material of this invention; Figure 4 a) Comparison of MC3T3-E1 cell viability after treatment with different concentrations of sodium bicarbonate nanoparticles, determined by the MTT assay; Figure 4 b) Comparison of MC3T3-E1 cell viability after treatment with different concentrations of PB NPs, determined by the MTT assay; Figure 4 c) is the control group, GelMA, Images of live / dead fluorescence staining of PB / GelMA and novel gel repair materials; Figure 4 d) Comparison of UV-Vis absorption spectra of different treatment groups; Figure 4 e) A statistical analysis comparison chart of hemolysis rates in different treatment groups; Figure 5 The figure shows the effect of the novel gel repair material of this invention on HUVEC tube formation and MC3T3-E1 cell migration behavior. Figure 5 a) A schematic diagram of the angiogenesis mechanism; Figure 5 b) This includes the control group and the GelMA group. PB / GelMA group and Images from angiogenesis experiments; Figure 5 c) Images of cell migration in the scratch assay at different time points; Figure 5 d) is a comparison chart of the quantitative analysis of the number of vascular nodes in different treatment groups; Figure 5 e) A statistical analysis and comparison chart of the wound closure percentages at 12 hours and 24 hours for different groups; Figure 6 This is a diagram showing the reactive oxygen species scavenging and pH adjustment mediated by the novel gel repair material of this invention; Figure 6 a) Fluorescence images of cells after different hydrogel treatments to detect oxidative stress levels using the DCFH-DA probe; Figure 6 b) Fluorescence images of the intracellular acidic microenvironment detected using the BCECF probe after different hydrogel treatments; Figure 6 c) A comparison chart of the quantitative analysis of the relative reactive oxygen species levels of each group under the corresponding conditions; Figure 6 d) is a comparison chart of the quantitative analysis of pH fluorescence intensity of each group under the corresponding conditions; Figure 7 This diagram illustrates the bone-inducing effects of different treatment groups on MC3T3-E1 cells according to the present invention. Figure 7 a) ALP staining image of MC3T3-E1 cells; Figure 7 b) ARS staining pattern of MC3T3-E1 cells; Figure 7 c) Comparison of OPN osteogenic marker expression in MC3T3-E1 cells of different treatment groups; Figure 7 d) Comparison of OCN osteogenic marker expression in MC3T3-E1 cells from different treatment groups; Figure 7 e) Comparison of RUNX2 osteogenic marker expression in MC3T3-E1 cells from different treatment groups; Figure 8 This is an evaluation diagram of the immunomodulatory properties of the novel gel repair material of the present invention; Figure 8 a) A schematic diagram showing how LPS induces RAW264.7 cells to polarize into M1 macrophages and IL-4 induces them to polarize into M2 macrophages; Figure 8 b) Comparison of relative expression levels of iNOS in RAW264.7 cells from different treatment groups; Figure 8 c) Comparison of relative expression levels of IL-1β in RAW264.7 cells from different treatment groups; Figure 8 d) Comparison of relative Arg-1 expression levels in RAW264.7 cells from different treatment groups; Figure 8 e) Comparison of relative expression levels of CD206 in RAW264.7 cells from different treatment groups; Figure 9 This is an in vivo osteogenic effect evaluation diagram of the novel gel repair material of this invention; Figure 9 a) Schematic diagram of the construction of the SD rat skull defect model; Figure 9 b) Representative microCT images of skull defects in rats 4 and 8 weeks after implantation of different hydrogels; Figure 9 c) Comparison of bone volume fraction in microCT evaluation results 4 and 8 weeks after implantation of different hydrogels; Figure 9 d) Comparison of bone mineral density in microCT assessment results 4 and 8 weeks after implantation of different hydrogels; Figure 9 e) Comparison of H&E staining of rat skull defects 4 and 8 weeks after implantation with different hydrogels; Figure 9 f) Comparison of Masson's trichrome staining of skull defects in rats 4 and 8 weeks after implantation with different hydrogels; Figure 10 Comparison of H&E staining results after 4 weeks of implantation of different hydrogels into different organs of SD rats; Figure 11Comparison of H&E staining results after 8 weeks of implantation of different hydrogels into different organs of SD rats; Figure 12 This is a scanning electron microscope image and elemental analysis diagram of the sodium bicarbonate nanomaterials before DSPE-PEG modification according to the present invention. Figure 13 This is a particle size distribution diagram of the sodium bicarbonate nanomaterials before DSPE-PEG modification according to the present invention; Figure 14 This is the UV-Vis-NIR II absorption spectrum of the PB NPs of this invention; Figure 15 This is a comparison chart of the degree of reaction at different time points in the decomposition of hydrogen peroxide by PB NPs to produce oxygen according to the present invention; Figure 16 Images of live / dead HUVEC cells after co-culturing in different groups. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 like Figure 1 As shown, this invention discloses a method for preparing a novel gel repair material, the technical solution of which includes the following steps: Step 1, Synthesis of sodium bicarbonate nanoparticles, 20 mg sodium oleate, 3 mL anhydrous ethanol, 1 mL oleylamine, and 10 mL n-hexane were added to a 20 mL glass bottle. After ultrasonic dispersion, 200 μL of ammonium bicarbonate aqueous solution (100 mg / mL) was added, and the mixture was stirred at room temperature for 10 min. The mixture was then centrifuged (11900 rpm) for 15 min and washed once with anhydrous ethanol. The elemental distribution of the resulting precipitate is shown below. Figure 12 As shown, its elemental distribution is uniform, through Figure 13 The particle size distribution diagram shows that the average particle size of the obtained sodium bicarbonate is approximately 200 nm; the obtained nano-sized sodium bicarbonate particles were dispersed in 1 mL of chloroform to obtain a sodium bicarbonate chloroform solution; Figure 12 The scale bar is 100 nm; Step 2, DSPE-PEG surface modification, Add 1 ml of DSPE-PEG chloroform solution (20 mg / mL) to 4 ml of sodium bicarbonate chloroform solution (one-quarter volume) prepared in step 1; then stir the mixture continuously at room temperature for 12 h, and then dry it under vacuum at 40 °C to obtain a white substance; The obtained white substance was verified by Fourier transform infrared spectroscopy and dynamic light scattering test. The test results are as follows: Figure 2 a, Figure 2 As shown in b, the white substance is DSPE-PEG modified sodium bicarbonate nanoparticles with a particle size of approximately 492 nm and no obvious aggregation. Step 3, Preparation of PB NPs 0.75 g of PVP and 0.0275 g of potassium ferricyanide were dissolved in 10 mL of 75% ethanol solution in the presence of 0.01 M HCl to obtain a reactive precursor solution. The reactive precursor solution was then placed in a water bath at 80 °C and reacted for 3 h before being cooled to room temperature. The resulting dark blue reaction mixture was washed three times with an ultrafiltration tube and freeze-dried for 24 h to obtain a dark blue substance. The obtained deep blue substance was subjected to dynamic light scattering and ultraviolet-visible-near-infrared spectroscopy tests, and the results are as follows: Figure 2 c. Figure 14 As shown, the dark blue substance obtained is PVP-modified PB NPs with a particle size of approximately 70 nm. Step 4, Preparation of composite gel repair material Weigh out solid GelMA, DSPE-PEG modified sodium bicarbonate nanoparticles obtained in step 2, and PVP-modified PB NPs obtained in step 3. Add all three to a PBS solution containing 0.25% w / v photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP). The final concentration of GelMA is 10%, the concentration of PB NPs is controlled at 50 μg / mL, and the concentration of sodium bicarbonate is 400 μg / mL. Irradiate with a UV flashlight light source at a wavelength of 405 nm for 2 minutes to cure GelMA and obtain the desired product. Composite hydrogels, such as Figure 2 As shown in d, this is a novel gel repair material.

[0025] To test the difference in gel strength produced by different GelMA concentrations, four final concentrations of GelMA (10%, 15%, 20%, and 25%) were prepared. After curing, their Young's modulus was measured, and the results are as follows: Figure 2As shown in Figure e, at a GelMA concentration of 10%, the hydrogel can provide sufficient mechanical strength to support skull defects while maintaining an environment conducive to cell adhesion. Therefore, a final concentration of 10% was selected as the baseline control. Further, at least one of DSPE-PEG modified sodium bicarbonate nanoparticles and PVP-modified PB NPs was added to the GelMA solution to obtain three groups of samples. The final concentration of GelMA in all three groups was controlled at 10%. After curing together with the pre-prepared 10% GelMA solution, three groups of composite hydrogel samples and one group of pure GelMA hydrogel samples were obtained. Mechanical properties, electron microscopy, swelling ratio, and degradation performance were tested on the four groups of hydrogel samples. The testing methods are as follows: Mechanical properties: Cylindrical hydrogel samples (10 mm in diameter and 3 mm in height) were prepared and Young's modulus was tested using a universal mechanical testing system (68SC type, Instron, Boston, USA). The modulus was calculated by the slope of the linear region of the stress-strain curve.

[0026] Scanning electron microscopy: The morphology of nanoparticles and nanocomposite hydrogels was observed using a transmission electron microscope (TECNAI G2 F20, FEI, USA) at an accelerating voltage of 30 kV. After the GelMA hydrogel and composite hydrogel were freeze-dried, platinum was sputtered at a current of 20 mA for 45 seconds, and the cross-sectional morphology of the samples was observed using a scanning electron microscope (SEM: Quanta 250, FEI, Hillsboro, Oregon, USA).

[0027] Swelling rate test: The swelling behavior of the composite hydrogel was evaluated by immersing the gel in 50 ml of PBS at ambient temperature for a certain period of time (weighed every two hours until equilibrium was reached). Before weighing, excess water on the surface of the gel was blotted dry with filter paper. After weighing, the gel was returned to the water for further swelling.

[0028] Formula for calculating swelling ratio: , in, This is the weight (g) of the sample before swelling. It is the weight (g) of the gel after swelling and soaking in water for time t.

[0029] Degradation performance test: The composite hydrogel was suspended in a neutral (pH 7.4) phosphate buffer solution, and the sample was incubated in a shaker incubator at 37℃. After wiping off the surface moisture at 0, 1, 2, 4, 6, 8, 10, 12, and 14 days, the mass loss was measured by weighing.

[0030] Test results are as follows Figure 2 As shown in f~i, by Figure 2As can be seen from f, the Young's modulus of the composite hydrogel samples is higher than that of the pure GelMA hydrogel samples, therefore they have sufficient mechanical strength; Figure 2 g indicates that The composite hydrogel possesses a porous microstructure, which facilitates the diffusion of oxygen, nutrients, and metabolic waste, as well as cell migration in three-dimensional space, highlighting its potential in bone regeneration; Figure 2 h indicates that The swelling ratio of the composite hydrogel was not significantly different from that of pure GelMA hydrogel. The moderate swelling property facilitates rapid absorption of surrounding tissue fluid, promotes the release of functional components from the nanoparticles, and adapts to morphological changes in bone defects, achieving close adhesion to the defect area. Figure 2 As can be seen, all hydrogel samples exhibited progressive degradation characteristics in the 14-day degradation test, among which... The degradation rate of the composite hydrogel is slightly higher than that of the pure GelMA hydrogel, but the overall degradation process is mild and controllable. Its degradation behavior matches the bone regeneration cycle and can maintain structural integrity and functional stability during the critical period of bone repair (2-4 weeks).

[0031] Furthermore, in order to analyze In the composite hydrogel, the antioxidant properties of PB NPs and the pH regulation ability of sodium bicarbonate nanoparticles were investigated. First, the free radical scavenging activity of PB NPs was evaluated by DPPH assay (the mechanism of action is as follows). Figure 3 (as shown in a).

[0032] Test method: DPPH powder was dissolved in anhydrous ethanol to a final concentration of 1 mM, and then a PB NPs solution (0.1 mg / mL) was prepared. Then, 300 μL of DPPH solution and 100 μL of sample were added to 2600 μL of ethanol, and the absorbance was measured at 517 nm at different time points. The initial antioxidant capacity was recorded as A0. The antioxidant capacity was recorded as At. The retention rate was calculated as At / A0*100%.

[0033] DPPH is a stable free radical with a strong absorption peak near 517 nm, such as Figure 3 As shown in b; the change in PBNPs clearance efficiency over time is as follows: Figure 3 As shown in Figure c, the scavenging efficiency increases significantly over time, rising sharply in the first 60 minutes and continuing to increase from 60 to 90 minutes. This indicates that PB NPs can neutralize DPPH free radicals through continuous electron transfer, producing a lasting antioxidant effect. This was further verified by ABTS free radical scavenging experiments (mechanism as follows). Figure 3 As shown in d), PB NPs are effective against water-soluble substances. It also demonstrates highly efficient removal capabilities, with removal efficiency gradually increasing over time (e.g.) Figure 3 As shown in e and f), this confirms its broad-spectrum scavenging activity against different types of free radicals; these results indicate that PB NPs can effectively scavenge DPPH free radicals in a time-dependent manner. To further explore its antioxidant function, this embodiment tested the ability of PB NPs to scavenge hydrogen peroxide, a major reactive oxygen species in vivo that catalyzes the generation of oxygen, reflecting catalase-like (CAT) activity.

[0034] Furthermore, the oxygen generation was tested using a portable dissolved oxygen meter to measure the oxygen concentration in the aqueous solution. After co-incubation with 10 mM hydrogen peroxide, its catalytic activity was evaluated at different time points. Subsequently, an oxygen probe (OXNP, Unisense A / S CO.LTD) was inserted to monitor the oxygen concentration in the solution in real time.

[0035] Dissolved oxygen monitoring showed that, compared with the PBS control group, PB NPs significantly increased oxygen levels, and this effect increased with increasing concentration, stabilizing around 50 μg / mL. Figure 3 g). Photographs taken at different time points further confirm the continuous generation of oxygen in the hydrogen peroxide solution (e.g. Figure 15 (As shown). These findings suggest that PB NPs alleviate oxidative stress by decomposing hydrogen peroxide and releasing oxygen, thereby creating a more favorable microenvironment for tissue repair.

[0036] Meanwhile, sodium bicarbonate is used as a safe and effective pH adjuster to combat the acidic environment commonly found in skull defects. Figure 3 As shown in h, with increasing sodium bicarbonate concentration, the pH gradually increased from approximately 6.0 to approximately 7.4, indicating its concentration-dependent buffering capacity. This process is accompanied by the release of sodium ions ( Figure 3 i) promotes the transport of bicarbonate ions to the defect area, neutralizes excess protons, and adjusts the local pH to a slightly alkaline range (approximately 7.2–7.4) most favorable to osteoblast activity. This buffering effect not only mitigates the inhibitory effect of acidity on osteoblasts but also inhibits the activity of osteoclast proton pumps, thereby reducing bone resorption and promoting conditions conducive to new bone formation.

[0037] Subsequent in vitro performance validation will be conducted, and the cell culture process involved in the validation is as follows: Cell culture medium composition: basal medium (89%), fetal bovine serum (10%), and penicillin-streptomycin (1%). Cells were cultured in an incubator at 37°C, 5% CO2, and 95% relative humidity, with the medium changed every three days. When MC3T3-E1 cells, RAW264.7 cells, and human umbilical vein endothelial cells (HUVECs) reached 80% confluence, they were passaged using trypsin digestion.

[0038] To assess the biocompatibility of the composite hydrogel material, cell viability was first detected using the MTT assay. Detection method: at 5%... MC3T3-E1 cells were cultured in a cell culture incubator in medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin). The intrinsic cytotoxicity of the material was assessed using an MTT assay. MC3T3-E1 cells were then... Cells were seeded at a density of [number] cells / mL into 96-well plates, and 0.1 mL of complete culture medium was added to each well. After incubation for 24 hours, the supernatant was removed, and culture medium containing different concentrations of sodium bicarbonate and PB NPs was added to the wells (incubated for 24 hours). The culture medium was aspirated, and then 10-20 μl of MTT was added to each well, and the cells were incubated for another 4 hours. After incubation, the MTT was carefully removed, and 100 μl of DMSO was added to each well. Cell viability was measured using a microplate reader (absorbance at 570 and 490 nm).

[0039] The results are as follows Figure 4 As shown in a and b, when the concentrations of PB NPs and sodium bicarbonate nanoparticles increased to 100 μg / mL, the cell viability remained above 80%, and no significant differences were observed between the groups. These results indicate that within this concentration range, both nanomaterials exhibit good cell compatibility and negligible toxicity, supporting cell survival and proliferation. Subsequently, in this embodiment, the nanoparticles were incorporated into GelMA hydrogel and photocrosslinked to prepare... PB / GelMA and PBS solution and pure GelMA hydrogel were used as control groups. The growth status of HUVEC and MC3T3-E1 cells co-cultured with the hydrogel was assessed using the calcein AM / PI live / dead staining method. Figure 4 c, Figure 16 The live / dead staining experiment procedure is as follows: Cells with the same cell density and MC3T3-E1 cells were seeded in 48-well plates. After overnight adhesion, the cells were co-incubated with the PBS-treated group and the drug-treated group at 37°C for 24 hours. Then, the cells were stained with AM / PI for 30 minutes, washed three times with PBS, and finally imaged by fluorescence microscopy (three replicates per group, for a total of eight samples).

[0040] Fluorescence imaging results showed that only weak PI-stained red fluorescence was detected in all groups, indicating very few dead cells, while strong green fluorescence predominated in the merged images, confirming high cell viability. These findings collectively indicate that the composite hydrogel provides a favorable microenvironment for cell survival and growth. Furthermore, hemolysis rate is a key indicator for assessing the blood compatibility of biomaterials, directly related to whether hemolytic reactions and vascular damage occur after material implantation. The hemolytic performance of each group of hydrogels was tested through in vitro hemolysis experiments. UV-Vis absorption spectroscopy (…) Figure 4 d) The results show that There was no significant difference in absorbance between the composite hydrogel, the PBS control group, and the pure GelMA hydrogel group. Quantitative analysis further confirmed this. Figure 4 (e) The hemolysis rates of all hydrogel groups were well below the safety threshold of 5%, indicating that they do not disrupt the integrity of red blood cell membranes upon contact with blood and exhibit excellent blood compatibility. Figure 4 c. The scale bar of Figure 16 is 50 μm.

[0041] In order to investigate The effect of composite hydrogels on in vitro angiogenesis; the formation of new blood vessels is closely related to local oxygen concentration (e.g., Figure 5 (as shown in a), for evaluation To explore the angiogenesis potential of the composite hydrogel, this embodiment first employs human umbilical vein endothelial cells (HUVECs) for in vitro angiogenesis experiments. The in vitro angiogenesis characterization method involves: 100 μL of HUVEC suspension (… Cells ( / mL) were seeded in Matrigel-coated 96-well plates. A material-free control group, a GelMA group, and a control group were set up. Group, PB / GelMA group and Group. Place the culture plate in an incubator and incubate for 4-6 hours to induce lumen formation. Take microscopic images and count the number of lumens.

[0042] Characterization results showed that the two oxygen-releasing groups (PB / GelMA and Both induced the assembly of tubular networks radiating from endothelial cell clusters, among which The resulting network structure was more extensive than that produced by using PB / GelMA alone. In contrast, the control group and the pure GelMA group produced only incomplete networks. Figure 5b). Quantitative analysis further confirmed that The group produced the most branch nodes and the longest main pipe length. Figure 5 d), indicating that continuous oxygen supply under hypoxic conditions can effectively promote the formation of vascular networks. This example then examined whether oxygen release affected the migration of osteoblast progenitor cells. MC3T3-E1 cells were used for a scratch assay. The experimental procedure was as follows: to test the effect of different materials and composite hydrogels on cell migration ability, 2 ml of cell suspension (concentration 1.5-2.0 composite hydrogel cells / mL) was seeded in 6-well plates, with separate control groups (no material), GelMA group, and... Group, PB / GelMA group and Group. The culture plates were placed in an incubator and cultured until the cell confluence reached 80%. Culture media containing different hydrogels were added, and a control group was set up. The wound healing status was observed under a microscope at 0h, 6h, 12h and 24h after drug addition.

[0043] The experimental results showed that, compared with the PBS control group and the GelMA group, The number of migrating cells in the group was nearly twice that of the previous group. Figure 5 (c, e). These findings indicate that prolonged oxygen release not only promotes endothelial tube formation but also enhances the migration of osteoblast precursors, both of which are crucial for coupling angiogenesis with bone regeneration. Figure 5 (The scale bars for b and c are 50 μm).

[0044] Osteoblasts originate from mesenchymal progenitor cells in the periosteum and bone marrow matrix. Mature osteoblasts form a monolayer on the bone surface, where they synthesize, secrete, and mineralize the extracellular matrix, playing a central role in bone formation and remodeling. However, hypoxia and excessive reactive oxygen species (ROS) impair osteoblast growth and differentiation while stimulating the proliferation and differentiation of osteoclast precursors. The resulting osteoclast surge leads to excessive secretion of acidic byproducts and hydrolytic enzymes, further exacerbating local acidosis. In this pathological cycle, restoring oxygen supply provides a promising strategy for reversing the imbalance between bone formation and resorption. To evaluate... The composite hydrogel's ability to modulate such harsh environments was demonstrated in this embodiment using hydrogen peroxide to establish an oxidative stress model, and different mediators were co-cultured with MC3T3-E1 cells. Figure 6 a) The control groups included the untreated NC group, the PBS control group, and the pure GelMA hydrogel group. Composite hydrogel group, PB / GelMA composite hydrogel group, Composite hydrogel assembly.

[0045] Intracellular reactive oxygen species (ROS) detection method: Intracellular ROS production was detected using a 2,7-dichlorofluorescein diacetate probe (DCFH-DA). MC3T3-E1 cells were cultured in an incubator containing 5% carbon dioxide at 37°C for 24 h. Cells were then stimulated with PBS or a drug treatment group and cultured for 4 h. The DCFH-DA probe was then added, and the cells were incubated at 37°C in the dark for 30 min. After washing with PBS, ROS levels were detected using a fluorescence microscope (ECLIPSE Ti2; Nikon, Japan). All images were acquired under the same conditions, and fluorescence intensity was quantitatively analyzed using ImageJ software (National Institutes of Health, USA).

[0046] Compared with the control group, cells treated with the composite hydrogel containing PB showed significantly reduced ROS levels, manifested as weaker green fluorescence, indicating effective relief of oxidative stress. Quantitative analysis further showed that intracellular ROS levels decreased to approximately 19% of the control group. Figure 6 c), reflecting the strong catalase-like activity and oxygen-releasing properties of the PB-containing hydrogel. Next, its buffering effect under acidic conditions was detected using the pH-sensitive probe BCECF. After culturing for 24 hours at pH 6.0, cells exposed to the sodium bicarbonate-containing hydrogel showed significantly reduced fluorescence compared to the control group or the PB-only hydrogel group. Figure 6 (b) confirmed that sodium bicarbonate still has an effective neutralizing effect on intracellular acidosis. Quantitative analysis ( Figure 6 d) Further verification showed that the sodium bicarbonate-containing composite hydrogel could significantly restore intracellular pH, thereby protecting osteoblasts from the adverse effects of an acidic microenvironment. Figure 6 The scale bars for a and b are 50 μm.

[0047] Bone formation involves two key stages: osteoblast differentiation and extracellular matrix mineralization. Alkaline phosphatase (ALP) and bone mineralization nodules (BMNs) are widely considered indicators of these processes. To assess... The role of composite hydrogels throughout the osteogenic cycle was demonstrated in this embodiment, including ALP staining for early differentiation and Alizarin Red staining for late mineralization. The experimental groups were consistent with the aforementioned MC3T3-E1 cell co-culture experiment, using the NC group, PBS control group, and pure GelMA hydrogel group. Composite hydrogel group, PB / GelMA composite hydrogel group, Composite hydrogel assembly.

[0048] Experimental method: MC3T3-E1 cells were seeded at the bottom of a culture plate. When the cell confluence reached 70%, osteogenic differentiation was induced using the OriCell® mouse MC3T3-E1 cell osteogenic differentiation kit. The differentiation medium was changed every three days.

[0049] (1) Alkaline phosphatase staining After 7 days of co-culture induction, cells were fixed with 4% paraformaldehyde for 30 minutes, and alkaline phosphatase activity was detected using an ALP staining kit. MC3T3-E1 cells were incubated with ALP chromogenic agent for 30 minutes under light-protected conditions, and the results were observed under a microscope.

[0050] (2) Alizarin Red staining Calcium nodules formed in cells were stained using an ARS staining kit. After 21 days of co-culture, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes, followed by 15 minutes of ARS staining. Calcium nodules were then observed using a camera microscope.

[0051] (3) qPCR (primer sequence) To investigate the roles of Runx2, OCN, and OPN genes in promoting osteogenic differentiation in materials, siRNAs targeting the mouse Runx2, OCN, and OPN genes were designed and synthesized. Before differentiation, cells were seeded in 6-well plates. When confluence reached 70%, the cells were co-cultured with various materials for osteogenic differentiation induction. The designed siRNAs are as follows: After 7 days, compared with the PBS control group, ALP staining in the group showed significant and stronger activity, indicating enhanced early osteogenic differentiation. Figure 7 a). By day 21, alizarin red staining confirmed more extensive calcium nodule formation in the hydrogel group, further supporting its ability to promote late-stage mineralization. Figure 7 b). These findings suggest that alleviating oxidative stress and maintaining oxygen supply under hypoxic conditions can steadily enhance osteogenic activity. This embodiment further examined the expression of key osteogenic markers, including osteopontin (OPN), osteocalcin (OCN), and the transcription factor Runx2. RT-qPCR analysis showed that these genes were upregulated in all hydrogel groups compared to the control group, among which… The group with the most significant effect (p<0.05 or p<0.01) Figure 7 These results collectively indicate that, Hydrogels promote osteogenic processes not only at the cellular level but also at the molecular level by coordinating reactive oxygen species scavenging, pH regulation, and sustained oxygen release. This synergistic regulation creates a favorable microenvironment for osteoblast differentiation and matrix mineralization, highlighting the powerful translational potential of this composite hydrogel in bone defect repair. Figure 7 In the mean squares, **p<0.01, ***p<0.001, and ****p<0.0001; Figure 7 (The scale bars for a and b are 250 μm). For evaluation Immunomodulatory effects of composite hydrogels: RAW 264.7 macrophages were co-cultured with hydrogels under different conditions for 24 hours, and then their polarization towards the M1 or M2 phenotype was induced by LPS and IL-4, respectively. Figure 8 a. The experimental group also used the NC group, PBS control group, and pure GelMA hydrogel group. Composite hydrogel group, PB / GelMA composite hydrogel group, (Composite hydrogel group). qRT-PCR detection results are as follows: Figure 8 As shown in the figure. Compared with the control group, The expression of M1 macrophage markers (iNOS and IL-1β) was significantly downregulated, while the expression of M2 macrophage markers (Arg-1 and CD206) was upregulated. These results indicate that the composite hydrogel can suppress pro-inflammatory phenotypes and promote anti-inflammatory phenotypes. In summary, these results demonstrate that... The composite hydrogel can effectively modulate macrophage polarization, inhibit M1-driven inflammatory responses, and promote M2-mediated repair, which helps create a more favorable immune microenvironment for bone regeneration. Figure 8 In the mean squares, **p<0.01, ***p<0.001, and ****p<0.0001. Subsequent in vivo experiments were conducted to validate the results, and all procedures were performed in accordance with the Chinese Guidelines for the Care and Use of Laboratory Animals. Six- to eight-week-old SD rats were purchased from Skebers Biotechnology Co., Ltd. Before and after surgery, the rats were housed in a specific pathogen-free (SPF) environment with free access to water and a standard rat diet.

[0052] based on The composite hydrogel exhibits excellent cell compatibility, suitable mechanical strength, osteogenic activity, and immunomodulatory capacity. This embodiment further utilizes a rat cranial bone defect model with a critical size defect (5 mm in diameter). Figure 9 a) Assess its in vivo performance.

[0053] Experimental Methods: Rats were individually caged for one week to acclimatize to their environment before surgery and were anesthetized with isoflurane inhalation. After incising the skin at the top of the head, a circular full-thickness bone defect with a diameter of 5 mm was prepared using a drill. Subsequently, pure GelMA hydrogel was implanted into the defect area of ​​mice in different groups, respectively. Composite hydrogels, PB / GelMA composite hydrogels Composite hydrogel was used, and a control group without any implanted materials was set up. The wound was sutured in layers. Penicillin was administered intramuscularly for three consecutive days postoperatively, and the incision was disinfected with povidone-iodine.

[0054] Micro-computed tomography analysis: Rats were sacrificed in two batches at 4 and 8 weeks post-surgery, and skull specimens were collected. The specimens were fixed using a 10% fixative. After formalin fixation, the regeneration status of the defect area was assessed using a microCT scanner (NMC-200). The three-dimensional structure of the cranial vault was reconstructed using Recon software, and the bone volume / tissue volume ratio (BV / TV) and bone mineral density (BMD) were calculated.

[0055] Histological evaluation: Specimens were decalcified with 0.5 M EDTA at room temperature for two weeks. After paraffin embedding, 5-degree decalcified sections were prepared and stained with hematoxylin-eosin (H&E) and Masson's solution. Images were acquired using a bright-field microscope (Zeiss Axiovert 200; Carl Zeiss, New York, USA).

[0056] Statistical analysis: Standardized data are indicated in the legend. All data are expressed as mean ± standard deviation. In the somatic imaging study, each group consisted of n=3 rats (e.g., bone volume fraction, trabecular thickness, trabecular number, relative gray value). Two-tailed unpaired t-tests were used to calculate significance (p-value).

[0057] Bone regeneration was monitored at 4 and 8 weeks post-implantation using micro-CT. Figure 9 As shown in b, all groups showed a progressive reduction in defect area over time, with new bone evolving from granular deposits in week 4 to a structure closer to the cortex in week 8. The control group (Con, PBS group) showed the slowest repair rate, while... The group exhibited the most extensive bone ingrowth and more mature tissue structure. Quantitative analysis further confirmed these trends. Bone volume fraction (BV / TV), Figure 9 c) Display It most effectively accelerated bone formation, showing a significant increase at 8 weeks compared to the control group (P<0.005). Similarly, Bone mineral density (BMD) of the composite hydrogel group Figure 9 d) is also the highest. These results collectively indicate that The composite hydrogel promotes the formation of denser mineralized tissue by simultaneously scavenging reactive oxygen species, continuously releasing oxygen, and buffering pH, thereby achieving robust bone regeneration in vivo. Histological staining results are consistent with micro-CT findings. H&E staining results ( Figure 9 e) shows that at 4 weeks post-surgery, The defective area in the control group showed a large number of osteoblasts, regular arrangement of newly formed bone trabeculae, and significant vascular infiltration; while the control group and the pure GelMA group were mainly composed of fibrous connective tissue, with few osteoblasts and insufficient angiogenesis. At 8 weeks post-surgery, In the composite hydrogel group, newly formed bone tissue exhibited a clear lacunar structure, with increased trabecular thickness and fusion to form a dense structure similar to cortical bone, with blurred boundaries with normal skull tissue. Other groups still showed abundant fibrous tissue, with sparse and disordered trabecular bone. Masson trichrome staining further revealed the synthesis and remodeling processes of the collagen matrix. Figure 9 f): 4 weeks post-surgery In the composite hydrogel group, abundant blue collagen fiber deposition was observed, forming a continuous matrix network around the bone trabeculae to support bone mineralization. At 8 weeks post-surgery, the collagen fibers were tightly bound to bone minerals, forming a mature bone tissue structure, while the collagen fibers in other groups were loosely distributed and showed lower levels of mineralization. These results indicate... The composite hydrogel can promote the synthesis of osteogenic matrix and tissue remodeling, providing a structural basis for the maturation of new bone. Finally, the in vivo biocompatibility of the hydrogel was evaluated. H&E staining was performed on major organs (heart, liver, spleen, lung, and kidney) at 4 and 8 weeks. Figure 10 , Figure 11 The histological features were normal, with no signs of inflammation or tissue damage, confirming the diagnosis. The composite hydrogel exhibits good biocompatibility. Figure 9 In the mean squares, **p<0.01, ***p<0.001, and ****p<0.0001; Figure 10 , Figure 11 The scale is 200 μm. The three-pronged synergistic repair strategy of "microenvironment reprogramming - immune regulation - osteogenic enhancement" proposed in this embodiment breaks through the limitations of the single function of traditional materials, and achieves simultaneous regulation of pathological microenvironment improvement and immune and osteogenic functions. It also reveals the synergistic regulatory effects of PB NPs' ROS scavenging and oxygen generation functions, and sodium bicarbonate's pH buffering function on macrophage polarization, elucidating the transcellular signal transduction mechanism of "physicochemical microenvironment - immune cells - osteoblasts". 1. First, there is the polarization regulation mediated by ROS scavenging: Prussian blue nanoparticles (PB NPs) have a highly efficient ROS scavenging ability and can directly capture ROS through a unique electron transfer mechanism. , These reactive oxygen species simultaneously decompose hydrogen peroxide to generate oxygen via catalase-like activity. This process can alleviate oxidative stress damage to macrophages and inhibit the activation of the NF-κB inflammatory signaling pathway—ROS, as a key upstream activator of this pathway, can block the transcriptional expression of M1 polarization-related genes (iNOS, IL-1β) by clearing them, thus creating conditions for M2 polarization. 2. Secondly, sodium bicarbonate plays a regulatory role in acid-base microenvironment homeostasis: through the precise neutralization reaction of bicarbonate ions and hydrogen ions, sodium bicarbonate regulates the acidic microenvironment (pH≈6.0) in the bone defect area to a physiologically suitable range (pH≈7.2-7.4). An acidic environment exacerbates M1 polarization by activating proton-sensitive channels within macrophages, while a stable pH environment upregulates the activity of the STAT6 signaling pathway in macrophages. This pathway is a core regulatory pathway for M2 polarization and promotes the expression of M2 markers such as IL-10 and Arg-1. 3. Assisted regulation of oxygen supply optimization: The local continuous oxygen supply generated by the decomposition of hydrogen peroxide by PB NPs can improve the metabolic state of macrophages. Hypoxia can induce macrophages to produce acidic products such as lactic acid through glycolysis, which can aggravate the inflammatory response. Sufficient oxygen supply can promote the recovery of macrophage mitochondrial function, enhance oxidative phosphorylation metabolism, provide energy support for M2 polarization, and inhibit hypoxia-inducible factor-1α (HIF-1α) mediated M1 polarization signaling. 4. Synergistic effect of material interface and cell interaction: The biomimetic extracellular matrix structure of gelma hydrogel provides a suitable scaffold for macrophage adhesion and growth. Its degradation products (gelatin derivatives) can be recognized by macrophages, activating intracellular anti-inflammatory signaling pathways. At the same time, the nanoscale effect and surface physicochemical properties of PB NPs can regulate the phagocytic behavior of macrophages, avoid inflammatory responses caused by excessive accumulation of material particles, and further enhance the M2 polarization tendency.

[0058] This embodiment provides a A composite hydrogel modulates the harsh microenvironment of bone defects by combining reactive oxygen species (ROS) scavenging, self-oxygenation, and pH buffering functions. By embedding Prussian blue nanoparticles (PBNPs) and sodium bicarbonate into a GelMA network, controlled oxygen release, sustained ROS scavenging, and effective neutralization of localized acidosis are achieved. These synergistic effects break the vicious cycle of hypoxia, oxidative stress, and acidosis, thereby enhancing osteoblast activity while inhibiting osteoclast overactivation. Furthermore, the tunable physicochemical properties of GelMA allow for customization for different defect morphologies and microenvironmental conditions, offering potential for personalized bone repair. In vivo studies further confirmed that this hydrogel significantly improved the defect microenvironment, promoted new bone formation, and significantly increased bone volume fraction and mineral density. These findings collectively indicate that… Composite hydrogels provide a multifunctional and clinically applicable platform for the repair of complex bone defects, and have great translational potential.

[0059] The materials used in this embodiment are from the following sources: Sodium oleate and methyl DSPE-PEG2000 were purchased from Anaiji Chemical Co., Ltd. Ammonium bicarbonate was purchased from Beijing Innocare Technology Co., Ltd. Polyvinylpyrrolidone (PVP) and potassium ferricyanide were purchased from Aladdin Biochemical Technology Co., Ltd. All chemical reagents used are of analytical grade and can be used directly without further purification.

[0060] DPPH and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Yuanye Biotechnology Co., Ltd. OriCell® mouse MC3T3-E1 cell osteogenic differentiation kit and Alizarin Red (ARS) staining kit were purchased from Cyagen Biosciences (China). BCECF-AM (pH fluorescent indicator probe), acridine orange (AO), ethidium bromide (EB), and alkaline phosphatase staining kit were purchased from Beyotime (China). OCN (osteocalcin), osteopontin (OPN), and Runx2 (Runt-related transcription factor 2) were purchased from Sangon Biotech Co., Ltd. Phosphate-buffered saline (PBS), (3-[4,5-dimethylthiazolyl-2-yl]-2,5-diphenyltetrazolium bromide) (MTT), and tetrahydrofuran (THF, anhydrous, 99.9%) were purchased from Sigma-Aldrich (Shanghai, China).

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a gel repair material, characterized in that, Includes the following steps: Step 1: Preparation of sodium bicarbonate nanoparticles. A chloroform solution for synthesizing sodium bicarbonate nanoparticles via microemulsion method; Step 2, modification of sodium bicarbonate nanoparticles, A chloroform solution of DSPE-PEG was added to a chloroform solution of sodium bicarbonate nanoparticles synthesized in step 1. The mixture was stirred and reacted at room temperature and then purified to obtain DSPE-PEG modified sodium bicarbonate nanoparticles. Step 3: Prepare PVP-modified PB NPs; Step 4: Prepare composite gel repair material. The DSPE-PEG modified sodium bicarbonate nanoparticles obtained in step 2, the PVP-modified PB NPs obtained in step 3, and 10% GelMA gel material were mixed to prepare a composite gel repair material. In the composite gel repair material, the concentration of PBNPs caused the oxygen release rate to reach a plateau, and the concentration of DSPE-PEG modified sodium bicarbonate nanoparticles was able to stabilize the pH of the composite gel repair material at 7.2-7.

4.

2. The method for preparing the gel repair material according to claim 1, characterized in that: In step 1, sodium oleate, anhydrous ethanol, oleylamine, and n-hexane are mixed and ultrasonically dispersed, then ammonium bicarbonate aqueous solution is added, and the mixture is stirred at room temperature to carry out the reaction. After the reaction is completed, the mixture is centrifuged, and the precipitate obtained by separation is washed with anhydrous ethanol. The precipitate is then dispersed in chloroform to obtain a chloroform solution of sodium bicarbonate nanoparticles.

3. The method for preparing the gel repair material according to claim 1, characterized in that: In step 2, the mixed solution is continuously stirred and reacted at room temperature for 12 h, and then vacuum dried at 40°C to obtain the product.

4. The method for preparing the gel repair material according to claim 1, characterized in that: In step 3, polyvinylpyrrolidone and potassium ferricyanide are dissolved in an ethanol solution containing HCl to obtain a reactive precursor solution. The reactive precursor solution is placed in a water bath at 80°C and reacted for 3 hours before being cooled to room temperature. The resulting dark blue reaction mixture is washed with an ultrafiltration tube and freeze-dried to obtain PVP-modified PB NPs.

5. The method for preparing the gel repair material according to claim 1, characterized in that: In step 4, GelMA, DSPE-PEG modified sodium bicarbonate nanoparticles, and PVP-modified PB NPs are added to a PBS solution containing a photoinitiator, and a composite gel is prepared by ultraviolet light irradiation.

6. A novel gel repair material, characterized in that: It is prepared by any one of the preparation methods described in claims 1-5.

7. The application of a novel gel repair material, characterized in that: The novel gel repair material as described in claim 6 is used for biological tissue repair.

8. The application of the gel repair material according to claim 7, characterized in that: The biological tissue repair includes bone tissue repair, which can construct an anti-inflammatory repair microenvironment and take into account both angiogenesis and osteogenic processes.

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