Cerium metal phenolic network hydrogel and method

Abstract

The invention belongs to the technical field of hydrogel, and particularly relates to cerium metal phenolic network hydrogel and a method. Comprising the following steps: respectively dissolving hyaluronic acid-phenylboronic acid and tannic acid in ultrapure water with the concentration of 20 mg mL, 40 mg mL and 20 mg mL; and mixing and stirring the solution, and then adding 30 [mu] L of 1M NaOH to start gelation to obtain the cerium metal phenolic network hydrogel. According to the hydrogel disclosed by the invention, the oxygen content, oxidative stress and inflammation are regulated by utilizing metal-polyphenol chemical synergism, and a promising dual-function platform is provided for intervertebral disc regeneration.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, specifically to a cerium metal phenolic network hydrogel and a method thereof. Background Technology

[0002] The description of the background art in this invention pertains to related technologies and is used merely for illustration and to facilitate understanding of the invention. It should not be construed as the applicant explicitly believing or presuming that the invention was prior art on the filing date of the first application.

[0003] Intervertebral disc degeneration (IVDD) is partly driven by hypoxia disruption, excessive reactive oxygen species (ROS), and chronic inflammation, ultimately leading to senescence of nucleus pulposus cells (NPCs) and degradation of the extracellular matrix (ECM). IVDD is a major cause of chronic low back pain, characterized by progressive loss of extracellular matrix (ECM) components in the central (IVD) nucleus pulposus (NP) region, accompanied by disruption of the normal hypoxic microenvironment. This altered oxygen tension interferes with the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, which maintains anaerobic metabolism and matrix synthesis in NP cells. Simultaneously, the accumulation of reactive oxygen species (ROS) and the imbalance in inflammatory cytokine expression further accelerate cellular senescence and apoptosis. In summary, these pathological factors create a harmful biochemical environment that leads to continuous tissue degradation and impairs regenerative potential.

[0004] Currently, IVDD treatment primarily focuses on symptom management rather than true regeneration. Non-surgical treatments such as physical rehabilitation, analgesics, or anti-inflammatory drugs can provide temporary relief but cannot restore intervertebral disc structure. Surgical methods such as spinal fusion and total disc replacement aim to restore mechanical stability but often lead to biomechanical alterations, adjacent segmental disease, and long-term complications. In recent years, regenerative strategies, including stem cell therapy, growth factor delivery, and gene editing, have gained attention. However, these interventions often fail in the harsh inflammatory and oxidative microenvironment of intervertebral disc degeneration, where poor cell survival and lack of mechanical support hinder lasting repair. Therefore, there is an urgent need for a multifunctional treatment system capable of simultaneously restoring biomechanical integrity and biochemical homeostasis. Summary of the Invention

[0005] The purpose of this invention is to provide a cerium metal phenolic network hydrogel and method. The hydrogel of this invention utilizes metal-polyphenol chemical synergy to regulate oxygen content, oxidative stress, and inflammation, providing a promising dual-functional platform for intervertebral disc regeneration.

[0006] The objective of this invention is achieved through the following technical solutions:

[0007] A method for preparing a cerium metal phenolic network hydrogel includes the following steps:

[0008] Hyaluronic acid-phenylboronic acid, tannic acid and Ce 3+ Solutions with concentrations of 15-20 mg / mL, 30-40 mg / mL, and 15-20 mg / mL were prepared separately, mixed, and then 1-1.2 M NaOH was added to initiate gelation to obtain a cerium metal phenolic network hydrogel; the hyaluronic acid-phenylboronic acid, tannic acid, and Ce... 3+ The molar ratio of NaOH to NaOH is 500-600:100-150:25-30:1.

[0009] Furthermore, the preparation of hyaluronic acid-phenylboronic acid includes the following steps:

[0010] Hyaluronic acid was dissolved in ultrapure water, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and n-hydroxysuccinimide were added; after stirring, 3-aminophenylboronic acid was added; after the reaction was completed, the product was purified by dialyzing and lyophilized; the molar ratio of hyaluronic acid, -COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, n-hydroxysuccinimide and 3-aminophenylboronic acid was 8-10:1:4-5:2-3:8-10.

[0011] A cerium metal phenolic network hydrogel, wherein the cerium metal phenolic network hydrogel is prepared by the method according to claim 1 or 2.

[0012] A performance testing method for cerium metal phenolic network hydrogels includes:

[0013] Characterization, self-healing, injectability and adhesiveness, dissolved oxygen measurement, in vitro free radical scavenging assay, cytotoxicity detection, hemolysis assay, in vitro antibacterial assay, intracellular ROS scavenging and anti-inflammatory properties, aging-related β-galactosidase staining and RT-qPCR, in vivo evaluation in rat IVDD model and in vivo evaluation in New Zealand white rabbit IVDD model.

[0014] Furthermore, the characterization of the cerium metal phenolic network hydrogel specifically includes the following steps: adding hyaluronic acid-phenylboronic acid-tannic acid and hyaluronic acid-phenylboronic acid-Ce... + Tannic acid-Ce + A cerium-metal phenolic network hydrogel was dropped onto a glass slide and dried at 60°C; Fourier transform infrared spectra were recorded on a Spectrum 3 spectrometer from 4000 to 400 cm⁻¹. 1 64 scans, resolution: 1cm 1 ;

[0015] The self-healing, injectability, and adhesion testing specifically included the following steps: Two hydrogel samples were used to observe self-healing, one stained pink with Rhodamine B; each hydrogel was cut in half and reconnected by placing the two halves of different colors at the contact point for 20-30 minutes; a cerium metal phenolic network hydrogel was loaded into a syringe and squeezed into PBS through a 24G needle to test injectability; the cerium metal phenolic network hydrogel was applied to a fractured, isolated rat spine and allowed to adhere for 5-10 minutes to assess adhesion, with the standard of adhesion being no detachment upon standing; all processes were recorded using a digital camera.

[0016] Dissolved oxygen measurement specifically includes the following steps: Place hydrogen peroxide solution in PBS (pH 6.0-6.5), add Ce... 3+ The reaction takes 16-20 minutes, during which the hydrogen peroxide and Ce react. 3+ The molar ratio was 1:3-4; dissolved oxygen was measured at multiple time points (0, 0.5, 1, 2, 4, 8, 12 and 16 minutes) using a portable oxygen meter.

[0017] Furthermore, the in vitro free radical scavenging assay specifically includes the following steps: The exudate of HPTC hydrogel is prepared under conditions of pH 6.0-7.5 and a concentration range of 2.5-40 mg / mL: HPTC hydrogel is mixed with PBS at a ratio of 5-10 ml / g, soaked at 37°C for 24-36 h, and the supernatant is collected and filtered sterilized to obtain the scavenging capacity of ABTS+·, DPPH·, and ·O2; ABTS and K2S2O8 are reacted in the dark at a molar ratio of 6-7:4-5 for 12-14 h to generate ABTS. + ·; The obtained ABTS + • Solution (OD) 734 =0.70±0.05) and concentrations of 2.5-20 mg / mL -1 The hydrogel secretions were mixed at a volume ratio of 1:1-1.5 and incubated in the dark at 23-25°C for 30-35 min. The absorbance at 734 nm was recorded on a microplate reader; the ABTS+ clearance rate was calculated using the following formula: × 100% In the formula, A0 is the absorbance of the ABTS+· reference, A1 is the absorbance of the sample, and A2 is the absorbance of the corresponding solvent reference. DPPH· removal assay: Add 1-1.2 mL of 0.15-0.2 mM DPPH·ethanol to the hydrogel exudate and incubate in the dark at 25°C for 30-35 min. Use DPPH· instead of ABTS· as a control, measure the absorbance at 517 nm, and calculate the removal efficiency. • Os2-caking experiment: Incubate the reaction mixture containing Tris HCl buffer, pyrogallol, and hydrogel secretions in the dark at 25°C for 5-10 min; the Tris HCl, pyrogallol, and hydrogel secretions... 2 The molar ratio of pyrogallol to hydrogel secretions was 28-30:4-5:6-7; absorbance values ​​at 325 nm were recorded at 0 min and 5 min. Oscavenging rate was calculated using the following formula: Where ΔA0 is the absorbance change of the pyrogallol control, ΔA s It is the change in the absorbance of the sample.

[0024] Furthermore, the cytotoxicity assay specifically includes the following steps: After incubating HUVECs, 3T3-L1 cells, and NPCs, the culture medium is replaced with an equal volume of fresh culture medium containing hydrogel exudate at a concentration of 0 to 200-250 mg / mL; followed by incubation with a solution containing calcein AM1-1.2 μg / mL. -1 The culture medium was replaced with a different medium; the fluorescence intensity at 520 nm was measured using an ELISA reader (excitation: 490 nm); cell viability was determined by comparing the fluorescence intensity of the treated samples with that of the untreated control group.

[0025] The hemolysis test specifically includes the following steps: Prepare a 10-12% red blood cell suspension using fresh rabbit blood; add HA-PBA, TA, and Ce to this suspension. 3+ Alternatively, place the samples in centrifuge tubes containing HPTC hydrogels; test tubes containing deionized water and PBS serve as positive and negative controls, respectively. After incubating at 37°C for 1-1.5 hours, centrifuge the samples at 3300-3500 rpm for 5-7 minutes. Record the absorbance of the supernatant at 540 nm using a microplate reader; calculate the hemolysis rate using the following formula:

[0026]

[0027] The in vitro antibacterial test includes the following steps: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii are selected, and the antibacterial activity of the hydrogel is evaluated using the plate count method; HA-PBA solution or HPTC hydrogel is placed in a centrifuge tube containing 2-2.5 mL of bacterial suspension; after incubation for 6-8 hours, the OD is recorded using a microplate reader. 600 The values ​​were determined, and bacterial survival was quantified on agar plates.

[0028] The intracellular ROS scavenging and anti-inflammatory properties assays included the following steps: RAW264.7 macrophages were cultured and stimulated with LPS; the control group received only culture medium; LPS-induced cells were incubated with HA-PBA, HPT, or HPTC hydrogels; cells were loaded with DCFH-DA and fluorescence was detected; ROS localization was visualized using CLSM; the expression of IL-1β, IL-4, IL-6, IL-10, and TNF-α was analyzed using RT-qPCR; total RNA was isolated using TRIzol, reverse transcribed, and amplified using SYBR Green premix; GAPDH was used as an endogenous control.

[0029] Furthermore, the senescence-related β-galactosidase staining and RT-qPCR assays specifically include the following steps: NP cells are cultured and incubated with 100-120 μM H2O2 to induce senescence; the control group receives fresh culture medium; the cells are then treated with HA-PBA, HPT, or HPTC hydrogels; senescence is assessed by β-galactosidase staining.

[0030] The expression of p16, p21, ERK2, HIF-1α and GLUT1 was quantified by RT-qPCR, with GAPDH as an internal control.

[0031] Further, the in vivo evaluation of the rat IVDD model specifically included the following steps: Male Sprague-Dawley rats were acclimatized under standard conditions for one week; five animals in each group were randomly divided into: control group (no puncture), PBS group (puncture, injection of 5 μL PBS), HA-PBA group (puncture, injection of 5 μL HA-PBA solution), HPT group (puncture, injection of 5 μL HPT mixed solution), and HPTC hydrogel group (puncture, injection of 5 μL HPTC hydrogel); under 3% isoflurane anesthesia, a 21g puncture needle (5mm depth) was used to percutaneously puncture the coccygeal intervertebral disc (Co3 / 4~Co7 / 8), rotating 360° for 30-60 seconds; immediately after needle removal, 10-15 μL of the corresponding preparation was injected into the nucleus pulposus using a 33-g microsyringe. Buprenorphine 0.05mg / kg SC was administered for analgesia for 36-48 hours postoperatively.

[0032] Lumbar intervertebral discs (IVDs) were examined using a digital X-ray imaging system (Kubtec Model XPERT.8, USA). Subsequently, the disc height index (DHI) was measured; magnetic resonance imaging (MRI) was performed; and T2-weighted MR images were acquired to assess the water content and structural health of the IVDs. Imaging parameters included a repetition time of 3500 ms and an echo time of 120 ms. The scan range was 200 × 200 mm, and the slice thickness was 1.4 mm to ensure detailed cross-sectional images. Quantitative analysis of the MRI scans included measuring the grayscale values ​​of NPs in different samples using ImageJ software.

[0033] Intervertebral disc specimens were obtained 4 or 8 weeks later, decalcified in 10% EDTA solution for 1.5-2 months, embedded in paraffin, and then cut into 5 μm thick sections for hematoxylin-eosin (He), saffron O-fast green (SO) staining, and immunohistochemical staining. The sections were imaged using a fluorescence microscope (ECLIPSE Ts2R, Nikon), and the intensity of immunohistochemical staining was semi-quantitatively analyzed using ImageJ software.

[0034] NP tissues from PBS and HPTC hydrogels were lysed with Trizol reagent and rapidly frozen in liquid nitrogen to stabilize RNA; RNA was extracted and its purity was determined; strand-specific libraries were prepared and sequenced; raw reads were trimmed, aligned to a reference genome using HISAT2, and counted; expression levels were expressed as the number of fragments per million sequencing fragments per thousand bases of transcript; differentially expressed genes were identified as DESeq2 (Q<0.05, |log2fold-change|≥1); DEGs were functionally enriched and visualized in R (v3.2.0).

[0035] Further, the in vivo evaluation of the New Zealand white rabbit IVDD model included the following steps: Male New Zealand white rabbits were anesthetized with ketamine / thiazide, and a minimally invasive paramedian approach was used to expose the L1-L6 lumbar vertebrae; an 18-gauge needle was inserted to induce partial rupture of the annulus fibrosus and remove the nucleus pulposus; the groups were divided into: control group (L1-2, no puncture), PBS group (L2-3, puncture and injection of 20-30 μL PBS), HA-PBA group (L3-4, puncture and injection of 20-30 μL HA-PBA solution), HPT group (L4-5, puncture and injection of 20-30 μL HPT mixed solution), and HPTC hydrogel group (L5-6, puncture and injection of 20-30 μL HPTC hydrogel); all injections were completed immediately after puncture. Buprenorphine 0.03 mg / kg SC was administered postoperatively for analgesia for 48-72 hours.

[0036] X-ray and MRI examinations were performed using the same method as for rat modeling. Intervertebral disc specimens were harvested at 4 and 8 weeks post-surgery for histological examination via grinding and sectioning. The intervertebral discs were cut into 1–2 mm thick slices and completely fixed with neutral formaldehyde buffer. The slices were then ground until the disc-like structure was clearly visible. The bone slices were transferred to sufficient water until the washings became clear, followed by H&E and F&O staining. The embodiments of this invention have the following beneficial effects:

[0037] The product of this invention passes TA-Ce 3 The formation of a dual dynamic cross-linked network of borate ester bonds and coordination bonds in the metal-polyphenol network (mpn) results in shear-thinning injectability, self-healing ability, tissue adhesion, and cell compatibility. In vitro, HPTC hydrogel exhibits broad-spectrum ROS scavenging activity, downregulates the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and upregulates anti-inflammatory markers (IL-4, IL-10). Importantly, Ce... 3 HPTC hydrogel effectively reduced dissolved oxygen levels (105% at 15 minutes, compared to 115% in the control group), promoted the restoration of HIF-1α signaling expression by HPTC hydrogel, and alleviated NPC aging under HO2 oxidative stress. In rat and rabbit IVDD models, a single minimally invasive injection maintained intervertebral disc height index and MRI signal intensity, enhanced aggregate protein and type II collagen deposition, inhibited inflammatory mediators, increased HIF-1α, and simultaneously reduced p21 in situ. Transcriptomic analysis further revealed the role of the HIF-1α pathway in ECM regeneration. HPTC hydrogel utilizes metal-polyphenol chemistry to synergistically regulate oxygen content, oxidative stress, and inflammation, providing a promising dual-functional platform for intervertebral disc regeneration. Attached Figure Description

[0038] Figure 1 (A) Schematic diagram of HPTC hydrogel preparation, (B) Signaling pathways for reduced NPC aging, and (C) The therapeutic mechanism of intervertebral disc regeneration in rats and rabbits.

[0039] Figure 2 Preparation and characterization of HPTC hydrogels. (A) Preparation mechanism, (B) Gelation process, and (C) Scanning electron microscopy images of the fracture surface of HPTC hydrogels. (D) Fourier transform infrared spectra of HP-Ce3+ mixtures, (E) HPT mixtures, (F) TA-Ce3+ mixtures, and (G) HPTC hydrogels. (H) G' and G" values ​​of HPTC hydrogels under strain scanning. (I) Images of self-healing behavior and (J) Alternating strain measurements of HPTC hydrogels. (K) Injection demonstration and (L) Viscosity-shear rate curves of HPTC hydrogels. (M) Adhesive properties of HPTC hydrogels.

[0040] Figure 3In vitro and in vivo biocompatibility of HPTC hydrogel. (A) Fluorescence images and (BD) cell viability of various cells cultured in exudates of HPTC hydrogels at different concentrations (B: HUVECs, C: 3T3-L1, and D: NPCs). Statistical analysis was performed by one-way ANOVA and Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=3). **P<0.01, ***P<0.001. (E) Photographs and hemolysis rates of erythrocytes treated with HPTC hydrogel or different components (Insert image: Photographs of erythrocytes treated with hydrogel or different components). Statistical analysis was performed by one-way ANOVA and Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=3). *P<0.05, ***P<0.001. (F) H&E-stained sections of major organs of rats two months after HPTC hydrogel injection into ganglia.

[0041] Figure 4 In vitro immunomodulatory activity of HPTC hydrogel. The free radical scavenging rates of (A) ABTS+, (B)·DPPH·, and (C)·O2- by exudates of different concentrations of HPTC hydrogel at pH 7.4 or 6.0. Error bars: mean ± standard deviation (n = 3). (D) Fluorescence microscopy images and (E) Quantitative analysis of reactive oxygen species levels in RAW-264.7 macrophages after different treatments (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n = 3), ***P < 0.001. Relative mRNA expression levels of (F) TNF-α, (G) IL-1β, (H) IL-6, (I) IL-4, and (J) IL-10 in RAW-264.7 macrophages after different treatments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=3). **P<0.01, ***P<0.001.

[0042] Figure 5The ability of the hydrogel HPTC to delay the aging process of neurons. (A) Measurement of dissolved oxygen content in hydrogen peroxide solution. (B) Oxygen content in hydrogen peroxide and hydrogen peroxide-Ce3+ solutions at different time points. (C) Two-stage decomposition process of hydrogen peroxide with and without Ce3+. (D) Neuronal images stained with SA-β-Gal and (E) Quantitative analysis of SA-β-Gal positive cells after different treatments. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=3), ***P<0.001. Relative mRNA expression levels of (F)p16, (G)p21, (H)ERK2, (I)HIF-1α, and (J)GLUT1 in neurons under different treatments. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001.

[0043] Figure 6 Evaluation of the therapeutic effect of HPTC hydrogel in a rat model of preoperative intervertebral disc degeneration. (A) Schematic diagram of HPTC hydrogel treatment in a rat model of intervertebral disc degeneration. (B) Representative X-ray images of the rat model of intervertebral disc degeneration at weeks 4 and 8, and (C) Quantitative analysis of DHI. Statistical analysis was performed using a two-way ANOVA followed by a Tukey post-hoc test. Data are expressed as mean ± standard deviation (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001. (D) Representative MRI images of the rat model of intervertebral disc degeneration at weeks 4 and 8, and (E) Quantitative analysis of Pfirrmann grading. Statistical analysis was performed using a two-way ANOVA followed by a Tukey post-hoc test. Data are expressed as mean ± standard deviation (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001. Results of (F)HE and (G)F&O staining in a rat intervertebral disc degenerative disease model at weeks 4 and 8.

[0044] Figure 7Immunohistochemical results of HPTC hydrogel in a rat model of preoperative intervertebral disc degeneration. (A) Immunohistochemical staining, (BE) Quantitative analysis of Aggrecan, Collagen II, IL-1β, and TNF-α in the rat model of intervertebral disc degeneration at week 8 (B: Aggrecan, C: Collagen II, D: IL-1β, E: TNF-α). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=5), *P<0.05, ***P<0.001. (F) Immunohistochemical staining and (G) Quantitative analysis of HIF-1α in the rat model of intervertebral disc degeneration at week 8. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n=5), ***P<0.001. (H) Immunohistochemical staining and (I) Quantitative analysis of p21 in a rat intervertebral disc degeneration model at week 8. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n = 5), ***P < 0.001.

[0045] Figure 8 Transcriptome sequencing analysis. (A) Transcriptome volcano plot for analyzing differentially expressed genes. (B) GO richness analysis of differentially expressed genes. (C) Heatmap containing all genes of interest. (D) KEGG analysis of differentially expressed genes. (E) Enrichment analysis of the GSEA set of KEGG-related pathways. Quantitative analysis of the contents of (F) hyaluronic acid, (G) collagen II, (H) IL-1β, (I) TNF-α, (J) HIF-1α, and (K) p21 in nanoparticles under different treatments. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001.

[0046] Figure 9The therapeutic effect of HPTC hydrogel in a rabbit model of intervertebral disc degeneration (IVDD) was evaluated. (A) Schematic diagram of HPTC hydrogel treatment in the rabbit IVDD model. (B) Representative X-ray images of the rabbit IVDD model at week 4 and week 8, and (C) Quantitative analysis of DHI. Statistical analysis was performed using a two-way ANOVA followed by a Tukey post-hoc test. Data are expressed as mean ± standard deviation (n = 5), ***P < 0.001. (D) Representative MRI images of the rabbit IVDD model at week 4 and week 8, and (E) Quantitative analysis of Pfirrmann grading. Statistical analysis was performed using a two-way ANOVA followed by a Tukey post-hoc test. Data are expressed as mean ± standard deviation (n = 5), ***P < 0.001. (F) H&E and (G) F&O staining of the rabbit IVDD model at week 4 and week 8. Detailed Implementation

[0047] The present application will be further described below with reference to the embodiments.

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, in the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Different embodiments can be substituted or combined, and for those skilled in the art, other implementation methods can be obtained based on these embodiments without creative effort.

[0049] Extensive research by the applicant has revealed that injectable hydrogel systems offer unique advantages in terms of mechanical support and biomodulation, making them a novel target for IVDD (intravascular discectomy). Due to their high water content and tunable viscoelasticity, hydrogels can mimic the mechanical properties of natural NP (non-vascular) tissue, buffering compressive loads while conformally filling irregularly shaped defects. Furthermore, their injectability allows for minimally invasive delivery, which is particularly beneficial for reaching the deep avascular disc core. In addition to physical repair, hydrogels can also serve as carriers for therapeutic drugs, such as anti-inflammatory agents, antioxidants, and even living cells, enabling direct, local, and sustained release within degenerated tissue. Importantly, the incorporation of dynamic cross-linking mechanisms endows hydrogels with desired properties, such as self-healing, shear thinning, and tissue adhesion, enhancing their suitability for use in the mechanically dynamic IVD environment.

[0050] Among these design strategies, integrating metal-polyphenol networks (MPNs) into hydrogel scaffolds offers a powerful approach to conferring multiple therapeutic functions. MPNs are formed through the coordination of metal ions with polyphenol ligands, creating supramolecular assemblies with diverse bioactivities. Tannic acid (TA), a plant-derived polyphenol, has attracted interest due to its inherent antioxidant, anti-inflammatory, and antibacterial properties. In previous studies, TA-based hydrogels have demonstrated the dual functionality of free radical scavenging and reversible covalent interactions, facilitating dynamic crosslinking with chitosan, phenylboronic acid, or collagen. Simultaneously, most metal ions in MPNs exhibit multiple functions and bioactivities. For example, Ce... + Using Ce 4+ Participating in redox cycles allows for the simultaneous scavenging of ROS (·OH, H2O2, etc.) and oxygen consumption, which helps to rebuild the hypoxic microenvironment crucial for NP cell survival and ECM synthesis. Therefore, incorporating TA-Ce mp3+n into hydrogels may yield multiple functions, promoting IVDD repair by modulating local oxygen content and mitigating oxidative damage.

[0051] Based on the above ideas, the applicant has developed a method based on hyaluronic acid-phenylboronic acid (HA-PBA) and TA-Ce 3 +mpn dual-crosslinked injectable hydrogel (HPTC hydrogel). The dynamic borate ester bond between HA-PBA and TA provides a self-healing, reversible network, while the synergy of Ce+ with TA introduces a secondary crosslinking mechanism, enhancing mechanical integrity and bioactivity. Figure 1 A). In vitro experiments have demonstrated that TA-Ce in HPTC hydrogel 3 +mpn not only scavenge free radicals and reduce the expression of inflammatory cytokines, but also restores the hypoxic niche and protects NP cells from senescence. Figure 1 B). Furthermore, animal studies in rat and rabbit IVDD models have confirmed that HPTC hydrogel can maintain intervertebral disc height and structure, promote ECM regeneration, upregulate HIF-1α signaling, and downregulate aging markers such as p21 (B). Figure 1 C). By synergistically modulating the intervertebral disc microenvironment, this biomaterial holds promise as a minimally invasive, multifactorial treatment platform to halt or reverse the progression of IVDD.

[0052] A method for preparing a cerium metal phenolic network hydrogel includes the following steps:

[0053] Hyaluronic acid-phenylboronic acid, tannic acid and Ce 3+Solutions with concentrations of 15-20 mg / mL, 30-40 mg / mL, and 15-20 mg / mL were prepared separately, mixed, and then 1-1.2 M NaOH was added to initiate gelation to obtain a cerium metal phenolic network hydrogel; the hyaluronic acid-phenylboronic acid, tannic acid, and Ce... 3+ The molar ratio of NaOH to NaOH is 500-600:100-150:25-30:1.

[0054] In some embodiments of the present invention, the preparation of hyaluronic acid-phenylboronic acid includes the following steps:

[0055] 1 g (2.5 mmol) of hyaluronic acid was dissolved in 100 mL of ultrapure water. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and n-hydroxysuccinimide were added in a molar ratio of -COOH: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: n-hydroxysuccinimide. After stirring, 3-aminophenylboronic acid was added. After the reaction was complete, the product was purified by dialyzing and then lyophilized.

[0056] A cerium metal phenolic network hydrogel, wherein the cerium metal phenolic network hydrogel is prepared by the above-described method.

[0057] A performance testing method for cerium metal phenolic network hydrogels includes:

[0058] Characterization, self-healing, injectability and adhesiveness, dissolved oxygen measurement, in vitro free radical scavenging assay, cytotoxicity detection, hemolysis assay, in vitro antibacterial assay, intracellular ROS scavenging and anti-inflammatory properties, aging-related β-galactosidase staining and RT-qPCR, in vivo evaluation in rat IVDD model and in vivo evaluation in New Zealand white rabbit IVDD model.

[0059] In some embodiments of the present invention, the characterization of the cerium metal phenolic network hydrogel specifically includes the following steps: mixing hyaluronic acid-phenylboronic acid-tannic acid and hyaluronic acid-phenylboronic acid-Ce... + Tannic acid-Ce + A cerium-metal phenolic network hydrogel was dropped onto a glass slide and dried at 60°C; Fourier transform infrared spectra were recorded on a Spectrum 3 spectrometer from 4000 to 400 cm⁻¹. 1 64 scans, resolution: 1cm 1 .

[0060] In some embodiments of the present invention, the self-healing, injectability, and adhesion testing specifically includes the following steps: using two hydrogel samples to observe self-healing, one of which is stained pink with rhodamine b; the hydrogel is cut in half and reconnected by placing the two halves of different colors at the contact point for 20 minutes; a cerium metal phenolic network hydrogel is loaded into a syringe and squeezed into PBS through a 24G needle to test injectability; the cerium metal phenolic network hydrogel is applied to a fractured isolated rat spine and allowed to adhere for 5 minutes to assess adhesion; all processes are recorded using a digital camera.

[0061] In some embodiments of the present invention, dissolved oxygen measurement specifically includes the following steps: using 64 mMcCe + Alternatively, PBS (pH 6.0) was treated with 16 mM H2O2 for 16 minutes. Dissolved oxygen was measured at multiple time points (0, 0.5, 1, 2, 4, 8, 12, and 16 minutes) using a portable oxygen meter (JPB-607A, Shanghai Lecai Instrument Co., Ltd., China).

[0062] In some embodiments of the present invention, the in vitro free radical scavenging test specifically includes the following steps: preparing exudate of HPTC hydrogel under conditions of pH 7.4 or pH 6.0 with a concentration range of 2.5-40 mg / mL to evaluate the scavenging ability of ABTS+·, DPPH·, and ·O2; reacting ABTS (7 mM) with K2S2O8 (5 mM) in the dark for 12 h to generate ABTS. + ·; The obtained ABTS + · Solution (OD) 734 =0.70±0.05) 1 mL of hydrogel secretions

[0063] (2.5-20 mg / mL) -1 Mix (pH 7.4 or 6.0) and incubate in the dark at 25°C for 30 min. Record the absorbance at 734 nm on a microplate reader; the ABTS+ clearance rate is calculated using the following formula:

[0064]

[0065] In the formula, A0 is the absorbance of ABTS+·reference, A1 is the absorbance of the sample, and A2 is the absorbance of the corresponding solvent reference.

[0066] DPPH clearance test: 5-40 mg / mL of hydrogel exudate was collected. -1 Add 1 mL of 0.15 mM DPPPH·ethanol (pH 7.4 or 6.0) and incubate in the dark at 25°C for 30 min. Use DPPH· instead of ABTS· as a control, measure the absorbance at 517 nm, and calculate the scavenging efficiency.

[0067] • Os-cavenging experiment: Prepare a solution containing 150 μL of 50 mM Tris-HCl buffer (pH 8.2), 20 μL of 25 mM pyrogallol, and 30 μL of hydrogel secretion (2.5-20 mg / mL). -1 The reaction mixture (pH 7.4 or 6.0) was incubated in the dark at 25°C for 5 min. The absorbance at 325 nm was recorded at 0 min and 5 min. The Oscavenging rate was calculated using the following formula: -

[0068]

[0069] Where ΔA0 is the absorbance change of the pyrogallol control, ΔA s It is the change in the absorbance of the sample.

[0070] In some embodiments of the present invention, the cytotoxicity assay specifically includes the following steps: HUVECs, 3T3-L1 cells, and NPCs are cultured at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells / well in 48-well plates; after 24 hours of incubation, the medium was replaced with an equal volume of fresh medium containing hydrogel exudate at a concentration of 0 to 200 mg / mL; after another 24 hours of incubation, the cells were seeded with calcein AM (1 μg / mL). -1 The culture medium was replaced with a culture medium containing ELISA; after 30 min, the fluorescence intensity at 520 nm was measured using an ELISA reader (excitation: 490 nm); cell viability was determined by comparing the fluorescence intensity of the treated sample and the untreated control group.

[0071] In some embodiments of the present invention, the hemolysis test specifically includes the following steps: preparing a 10% red blood cell suspension using fresh rabbit blood and diluting it with PBS; adding HA-PBA, TA, and Ce to the suspension. 3+ Alternatively, place the samples in centrifuge tubes containing HPTC hydrogels; tubes containing deionized water and PBS serve as positive and negative controls, respectively. After incubating at 37°C for 1 hour, centrifuge at 3,500 rpm for 5 minutes. Record the absorbance of the supernatant at 540 nm using a microplate reader; calculate the hemolysis rate using the following formula:

[0072]

[0073] In some embodiments of the present invention, the in vitro antibacterial test specifically includes the following steps: selecting Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii, and evaluating the antibacterial activity of the hydrogel using the plate count method. HA-PBA solution (20 mg / mL) is then added. -1 ) or HPTC hydrogel (100mg) was placed in a bacterial suspension containing 2mL of 1.0×10 7 CFUmL-1 The OD values ​​were collected in centrifuge tubes and cultured for 6 hours. OD values ​​were then recorded using a microplate reader. 600 The values ​​were determined, and bacterial survival was quantified on agar plates.

[0074] In some embodiments of the present invention, the intracellular ROS scavenging and anti-inflammatory properties assay specifically includes the following steps: RAW264.7 macrophages were cultured for 24 h and then stimulated with LPS; the control group received only culture medium; LPS-induced cells were incubated with HA-PBA, HPT, or HPTC hydrogels; cells were loaded with DCFH-DA and fluorescence was detected; ROS localization was visualized using CLSM; the expression of IL-1β, IL-4, IL-6, IL-10, and TNF-α was analyzed using RT-qPCR; total RNA was isolated using TRIzol, reverse transcribed, and amplified using SYBR Green premix; primer sequences (Table S1) were synthesized by Shan Ya (Shanghai, China). GAPDH was used as an endogenous control.

[0075] Table S1. Primers for RT-qPCR.

[0076]

[0077] In some embodiments of the present invention, the senescence-related β-galactosidase staining and RT-qPCR assay specifically includes the following steps: NP cells are cultured and incubated with 100 μM h2O2 to induce senescence; the control group receives fresh culture medium; the cells are then treated with HA-PBA, HPT, or HPTC hydrogels; senescence is assessed by β-galactosidase staining; the expression of p16, p21, ERK2, HIF-1α, and GLUT1 is quantified by RT-qPCR, and the primer set is shown in Table S2; GAPDH is used as an internal control.

[0078] Table S2. Primers for RT-qPCR.

[0079]

[0080] In some embodiments of the present invention, the in vivo evaluation of the rat IVDD model specifically includes the following steps: Male Sprague-Dawley rats were acclimatized under standard conditions for one week; five animals in each group were randomly divided into: control group (no puncture), PBS group (puncture, injection of 5 μL PBS), HA-PBA group (puncture, injection of 5 μL HA-PBA solution), HPT group (puncture, injection of 5 μL HPT mixed solution); HPTC hydrogel group (puncture, injection of 5 μL HPTC hydrogel); under 3% isoflurane anesthesia, a 21g puncture needle (5mm depth) was used to percutaneously puncture the coccygeal intervertebral disc (Co3 / 4~Co7 / 8), rotating 360° for 30 seconds. Immediately after needle removal, 10 μL of the corresponding preparation was injected into the nucleus pulposus using a 33-g microsyringe. Buprenorphine 0.05mg / kg SC was administered for analgesia for 48 hours postoperatively.

[0081] X-ray examination of the lumbar intervertebral disc (IVD) was performed using a digital X-ray imaging system (Kubtec Model XPERT.8, USA). The disc height index (DHI) was then measured using the established three-midline method with ImageJ software. Magnetic resonance imaging (MRI) was performed using a high-field 3.0T MRI system (Philips Intera Achieva 3.0T MR, USA). T2-weighted MR images were acquired to assess the water content and structural health of the IVD. Imaging parameters included a repetition time of 3500 ms and an echo time of 120 ms.

[0082] The scan range was 200 × 200 mm with a slice thickness of 1.4 mm to ensure detailed cross-sectional images. Quantitative analysis of the MRI scans included measuring the grayscale values ​​of NPs in different samples using ImageJ software.

[0083] Intervertebral disc specimens were obtained 4 or 8 weeks later, decalcified in 10% EDTA solution for 2 months, embedded in paraffin, and then cut into 5 μm thick sections for hematoxylin-eosin (He), saffron O-fast green (SO) staining, and immunohistochemical staining. The sections were imaged using a fluorescence microscope (ECLIPSE Ts2R, Nikon), and the intensity of the immunohistochemical staining was semi-quantitatively analyzed using ImageJ software.

[0084] In addition, NP tissues from PBS and HPTC hydrogels were lysed with Trizol reagent and immediately rapidly frozen in liquid nitrogen to stabilize RNA. RNA was extracted and its purity was determined using a NanoDrop spectrophotometer. Strand-specific libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform. Raw reads were trimmed using FASTP, aligned to a reference genome using HISAT2, and counted using HTSeq. Expression levels were expressed as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes

[0085] (DEGs) were identified as DESeq2 (Q<0.05, |log2fold-change|≥1). Functional enrichment of DEGs was performed in R (v3.2.0) using Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Reactome database, and key terms were visualized using ggplot2. Furthermore, we performed gene set enrichment analysis (GSEA) to reveal pathway-level changes.

[0086] In some embodiments of the present invention, the in vivo evaluation of the New Zealand white rabbit IVDD model specifically includes the following steps: male New Zealand white rabbits are anesthetized with ketamine / thiazide, and the L1-L6 lumbar vertebrae are exposed using a minimally invasive paramedian approach; an 18-gauge needle is inserted to induce partial rupture of the annulus fibrosus and the nucleus pulposus is removed; the groups are: control group (L1-2, no puncture), PBS group (L2-3, puncture and injection of 20 μL PBS), HA-PBA group (L3-4, puncture and injection of 20 μL HA-PBA solution), and HPT group (L4-5, puncture and injection of 20 μL HPT mixed solution).

[0087] HPTC hydrogel group (L5-6, 20μL HPTC hydrogel injected via puncture). All injections were completed immediately after puncture. Buprenorphine 0.03mg / kg SC was administered for analgesia for 72 hours postoperatively.

[0088] X-ray and MRI examinations were performed using the same methods as for rat modeling. Intervertebral disc specimens were harvested at 4 and 8 weeks for histological examination via grinding and sectioning. The discs were cut into 1–2 mm thick slices and completely fixed with neutral formaldehyde buffer. The slices were ground until the discoidal structure was clearly visible, then grinding was stopped at the appropriate time. The bone slices were carefully transferred to sufficient water until the washings became clear, followed by H&E and F&O staining.

[0089] result:

[0090] Preparation and characterization of HPTC hydrogels

[0091] The reduced ECM synthesis, redox homeostasis imbalance, and elevated oxygen levels observed in degenerated NP tissues provide important clues for hydrogel design. The preparation of HPTC hydrogels is achieved through a step involving the reaction of TA and Ce+ with HA-PBA under deprotonated conditions to form mpn, thus forming the HPTC hydrogel. Figure 2 A) Hyaluronic acid and 3-aminophenylboronic acid (3-PBA) were amination reaction to obtain a hyaluronic acid-phenylboronic acid conjugate (HA-PBA). Proton NMR spectroscopy confirmed successful grafting of PBA, with a grafting efficiency of 36.25%. Then, HA-PBA was combined with TA-Ce... 3+ mpn mixing, rapid gelation induced by pH adjustment and stirring ( Figure 2 B). Given the crucial role of hydrogel pore size in drug release, degradation, and interaction with cells or tissues, we examined the cross-sectional morphology of the hydrogel using scanning electron microscopy (SEM). Figure 3 As shown in Figure C, the HPTC hydrogel exhibits a porous structure with pore sizes between 50 and 80 μm, which is beneficial for substance exchange and cell infiltration.

[0092] Furthermore, Fourier transform infrared (FTIR) spectroscopy is used to study molecular interactions and cross-linking mechanisms. In HA-PBA-Ce... 3 +(HP-Ce 3 In the hp-3+ system, compared with HA-PBA, no contractile or stretching vibrations of the major functional groups were observed in the FTIR spectrum of the hp-3+ mixture. Figure 3 D) indicates that the interaction between HA-PBA and Ce is minimal. In the HA-PBA-TA (HPT) system, the attenuation of the toxic development peak at 3411 cm⁻¹ (TA's -OH) and the enhancement of the toxic development peak at 3443 cm⁻¹ (HA-PBA's -OH) indicate the borate bond interaction between the phenolic hydroxyl group and the phenylboronic acid group. Figure 2 E). Similarly, as Figure 3 As shown in F, TA-ce 3 +3411cm in the mixture -1 Peak decay (from the -OH group of TA) conforms to TA-ce 3 The formation of coordination bands in +mpn. These spectral changes were also observed in HPTC hydrogels. Figure 2 G) confirmed the borate bond and TA-Ce 3 Coordination coexistence is a dual physical cross-linking mechanism that stabilizes the hydrogel network.

[0093] In order to inject NPs into the disk, the hydrogel must exhibit good rheological properties. For example... Figure 2As shown in Figure H, within the shear strain range of 0.01–100%, the storage modulus (G′) of the HPTC hydrogel consistently exceeds the loss modulus (G′), reaching approximately 230 Pa, indicating a stable and flexible viscoelastic profile. Furthermore, its self-healing properties and injectability offer significant advantages. The injectability and self-healing properties of the HPTC hydrogel were verified using rheological and macroscopic methods. Figure 2 As shown in Figure I, different colors are...

[0094] After cutting and touching the HPTC hydrogel block, incubation at 25°C for 30 minutes resulted in the formation of a new hydrogel. This new hydrogel could be lifted with tweezers and did not detach when inverted. Self-healing properties were also demonstrated by incubating at a fixed frequency of 10 rads. -1 The alternating strain scanning test was used for evaluation. For example... Figure 3 As shown in Figure J, the hydrogel maintains an intact network under low shear strain (1%). However, this state is disrupted at high strain (300%), with the G″ value exceeding G'. Notably, after three alternating strains, the hydrogel's...

[0095] G′ returns to its initial level, indicating that network reconstruction is rapid due to borate bonds and TA-Ce. 3 The dynamic physical cross-linking network formed by coordination bonds gives HPTC hydrogels self-healing capabilities.

[0096] Furthermore, injectable hydrogels can be delivered to closed sites of the intervertebral disc in a minimally invasive manner, avoiding excessive damage to the disc caused by open wounds. Figure 3 As shown in K, the HPTC hydrogel can be smoothly injected into water with a syringe, demonstrating its injectability. Shear thinning tests further confirm the injectability of the HPTC hydrogel; its viscosity decreases with increasing shear rate. Figure 3 The result (L) indicates that the hydrogel exhibits fluidity under high shear stress. Considering the compressive stress of IVD, the adhesive properties of the hydrogel are crucial for achieving a durable therapeutic effect. In an ex vivo model, the rat spine was transected, and then HPTC hydrogel was injected into the lesion site. The hydrogel effectively bonded the two severed spinal segments (L). Figure 3 The HPTC hydrogel (M) exhibits significant tissue adhesion. This strong affinity from mussel-like biocatechins derived from TA promises to retain HPTC at the injection site, preventing leakage into the intervertebral disc space and thus reducing the frequency of repeated administration. These results demonstrate that HPTC hydrogels possess multifunctional properties, including self-healing, injectability, and adhesion, making them well-suited for the IVD environment.

[0097] In vitro and in vivo biocompatibility of HPTC hydrogels

[0098] High biocompatibility is essential for injectable hydrogel therapy of degenerative non-reproductive cells (NPs) in IVD to prevent adverse immune responses. The in vitro and in vivo biocompatibility of HPTC hydrogels was evaluated using cell compatibility, blood compatibility, and tissue compatibility. HUVECs, 3T3-L1 cells, and NPCs were used as typical cell models, and cell compatibility was assessed by incubating them with different concentrations of hydrogel exudate for 24 hours. Figure 3 As shown in Figure A, fluorescence microscopy revealed that live cells (stained green) maintained normal morphology and were present in numbers comparable to those at 0 mg mL⁻¹. However, at concentrations of 150 and 200 mg mL⁻¹, the reduction in live cells was significantly greater than at the practically applied concentrations. Figure 4 A). Quantitative viability assays confirmed that cell viability decreased continuously with increasing hydrogel exudate concentration, but most remained above or close to 75% (A). Figure 4 B) indicates that HPTC hydrogel has good cell compatibility.

[0099] To assess the blood compatibility of HPTC hydrogel, we performed a hemolysis test, as hemolysis rate is a key indicator of biocompatibility. Red blood cells were incubated with HPTC hydrogel or different components thereof for 1 hour. Figure 3 As shown in E, HA-PBA, TA, Ce 3 The hemolysis rates in both the + and HPTC hydrogel groups were less than 5%, but Ce 3 + The treated erythrocyte supernatant was pale red, indicating good blood compatibility. Furthermore, at 2 months, histological examination of the major organs, including the heart, liver, spleen, lungs, and kidneys, of rats treated with HA-PBA, HPT, and HPTC hydrogels and control animals revealed no pathological abnormalities. Figure 3 F). These results highlight the systemic biocompatibility of HPTC hydrogels, which can be attributed to their clinically relevant and biocompatible components: hyaluronic acid and TA are natural products and clinically used pharmaceuticals with well-known biocompatibility, while cerium nitrate (III) is a raw material for the synthesis of mof biomaterials for biomedical applications. These in vitro and in vivo results confirm the good biocompatibility of HPTC hydrogels and demonstrate their potential for biomedical applications.

[0100] Preventing post-injection bacterial contamination is equally important. HAPBA and HPTC hydrogels were co-cultured with four clinically relevant pathogens: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas baumannii. The turbidity (OD) of the culture medium was measured. 60 The antibacterial activity was assessed using OD and colony-forming units (CFU) on agar plates. As shown in Figures S2A-D, the OD values ​​of the two groups... 60The values ​​were all significantly reduced, indicating a marked decrease in bacterial density. Plate counting confirmed these findings, with hydrogel-treated cultures showing sparse colonies, while the untreated control group showed dense bacterial growth (Figure S2E). Quantitative analysis further showed that the TA-Ce mpns hydrogel containing HPTC achieved an inhibition rate of 90% against all four pathogens, while the inhibition rate of HAPBA hydrogel alone was only 55-90% (Figure S2F-I). These results indicate that the boric acid groups in HAPBA are insufficient to achieve superior antibacterial properties, while the incorporation of natural antibacterial tannins significantly enhances the bactericidal efficacy of the hydrogel, thereby reducing the risk of injection-induced infections.

[0101] In vitro antioxidant and anti-inflammatory activities of HPTC hydrogel

[0102] Balanced oxidative stress is crucial for a normal IVD environment, but degenerative intervertebral discs (NPs) induce excessive oxidation, triggering severe inflammatory responses in immune cells, potentially leading to an acidic microenvironment and elevated levels of reactive oxygen species (ROS) and inflammatory factors. Therefore, hydrogels with strong ROS scavenging properties can significantly delay intervertebral disc degeneration. This article first evaluates the antioxidant activity of HPTC hydrogels using a free radical scavenging assay. Figure 4 As shown, with decreasing concentration of HPTC hydrogel extraction and pH value, the free radical scavenging activity of the hydrogel gradually increased to 60-80% with increasing DPPH··O2- concentration, although no significant pH-dependent absorptivity was observed. + • Clearance response is observed. Previous studies have shown that TA-based MPNs exhibit pH-responsive dissociation and release behavior. Therefore, TA-Ce MPNs inherit this property, enabling them to respond to the acidic microenvironment of the degenerated nucleus pulposus by accelerating dissociation and cargo release. This facilitates faster ROS clearance, thereby improving their in situ therapeutic efficacy.

[0103] Subsequently, oxidative stress was induced in RAW 264.7 macrophages via lipopolysaccharide (LPS), followed by treatment with HA-PBA, HPT, and HPTC hydrogels to assess intracellular ROS scavenging capacity in vitro. Intracellular ROS levels were detected using the ROS-sensitive fluorescent probe DCFH-DA. Figure 4As shown in Figure D, ROS was stained with green fluorescence. Compared with the control group, macrophages in the LPS and HA-PBA groups showed obvious green fluorescence covering blue fluorescence (nucleus), indicating that these macrophages were under oxidative stress and had higher ROS levels. In contrast, macrophages treated with HPT and HPTC hydrogels showed minimal green fluorescence, indicating lower ROS levels in these macrophages. Quantitative analysis of ROS fluorescence intensity confirmed these observations; the mean fluorescence intensity of the LPS and HA-PBA groups was significantly higher than that of the control group, HPT group, and HPTC hydrogel group. Figure 6 E). Furthermore, the average fluorescence intensity of the three groups was similar, with no significant difference. These results indicate that TA plays a crucial role in free radical scavenging in the HPTC hydrogel system, thereby significantly alleviating IVDD.

[0104] Besides the challenges posed by oxidative stress, dysregulated inflammation significantly promotes IVDD, and macrophages play a crucial role in this inflammatory response. To explore the anti-inflammatory properties of HPTC hydrogel, we induced an inflammatory state by stimulating RAW264.7 macrophages with LPS. Subsequently, they were incubated with HA-PBA, HPT, and HPTC hydrogels, and the levels of inflammatory cytokines TNF-IL-1, IL-4, IL-6, and IL-10 were measured. Figure 4 As shown in the FH, compared with the LPS and HA-PBA groups, the expression levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were significantly reduced in macrophages treated with HPT or HPTC hydrogels, while their expression levels were significantly increased compared with the untreated control group. Notably, the groups containing TA (HPT and HPTC hydrogels) also showed a significant upregulation of anti-inflammatory cytokines such as IL-4 and IL-10. Figure 4 These findings suggest that TA-Cempn can modulate the inflammatory cytokine profile of macrophages, providing a potential strategy for suppressing the inflammatory cascade associated with IVDD.

[0105] HPTC hydrogel's ability to alleviate NPPC aging

[0106] In IVDD, excessive oxidative stress and inflammation lead to increased local oxygen levels, such as the release of oxygen from the decomposition of hydrogen peroxide. This oxygen-rich environment may be detrimental to NPPCs, as they are physiologically adapted to hypoxic conditions. Therefore, reducing excessive oxygen accumulation is beneficial for promoting intervertebral disc repair. To verify the ability of Ce+ ions in HPTC hydrogel to reduce oxygen concentration, the hydrogel was co-incubated with hydrogen peroxide, and the dissolved oxygen content in the mixture was measured at different time points using a dissolved oxygen meter. Figure 5 A). For example Figure 5As shown in Figure B, without treatment, hydrogen peroxide undergoes spontaneous decomposition, leading to a gradual increase in dissolved oxygen concentration over time. In contrast, the addition of Ce... 3+ Afterwards, although dissolved oxygen still increased slightly, the oxygen concentration after 15 minutes only reached 105% of the initial level, significantly lower than the untreated hydrogen peroxide group (115%). This indicates that Ce+ ions effectively reduced oxygen accumulation by promoting the catalytic conversion of reactive oxygen species. Specifically, in the pathological microenvironment of IVDD, oxygen-containing free radicals react to generate H2O2, which subsequently decomposes and leads to an increase in oxygen levels (stage 1). Figure 5 C). However, Ce + It can undergo a redox reaction with hydrogen peroxide, preventing the increase of molecular oxygen (Phase 2). Figure 5 C).

[0107] To further evaluate the ability of HPTC hydrogel to alleviate hydrogen peroxide-induced cellular senescence, npc was co-incubated with hydrogen peroxide, followed by treatment with HA-PBA, HPT, or HPTC hydrogel. Cellular senescence was assessed by staining for senescence-associated β-galactosidase (SA-β-Gal) and the expression of senescence-associated markers p16 and p21. Figure 5 As shown in Figure D, extensive blue staining was observed in the H2O2 and HA-PBA groups, indicating high β-galactosidase activity and a marker of cellular senescence. In contrast, NPCs treated with ta-containing hydrogels, particularly HPTC hydrogels, showed minimal blue staining, indicating reduced senescence. This effect is attributed to the presence of Ce+ ions, which may suppress oxidative stress and its downstream effects. Quantification of SA-β-Gal positive cells further supports these findings, indicating a significantly higher percentage of senescent cells in the H2O2 and HA-PBA groups compared to the HPT and HPTC groups, with no significant difference between the HPTC group and the untreated control group. Figure 5 E). Furthermore, the expression of aging-related biomarkers p16 and p21 showed a similar trend; HPT and HPTC hydrogels significantly downregulated h2O2-induced overexpression of p16 and p21 genes, with no significant difference compared to the control group. Figure 5 F, G).

[0108] Furthermore, excessive oxygen within the intervertebral disc can reduce or inactivate the hypoxia-inducible factor (HIF) signaling cascade, disrupting glycolysis, extracellular matrix synthesis, and other essential cellular processes, thereby accelerating nasopharyngeal carcinoma senescence. Therefore, we quantified the transcriptional levels of HIF-1α and its pathway genes. Figure 5 As shown in HJ, npc treated with HPT or HPTC hydrogels exhibited significant upregulation of HIF-1α, upstream kinase ERK2, and downstream glycolytic transporter GLUT1, including Ce. 3The hydrogel produced the greatest enhancement of HPTC. In contrast, the hydrogen peroxide group and the HA-PBA group showed reduced or at-baseline expression compared to the untreated control group. These data suggest that the oxygen-rich environment generated by free radical decomposition inhibits HIF-1α, which in turn reduces GLUT1-mediated glycolysis, impairs cellular energy metabolism, and drives senescence; a negative feedback loop then further downregulates the upstream regulator ERK2.

[0109] In summary, these findings demonstrate that TA-Ce+mpn in HPTC hydrogel can effectively scavenge reactive oxygen species and alleviate hyperoxia-induced cellular senescence by restoring the expression of the HIF signaling pathway. Among these components, Ce+ plays a crucial role in driving these protective effects. These results highlight the therapeutic potential of HPTC hydrogel for IVDD.

[0110] In vivo therapeutic effects of HPTC hydrogel on rat IVDD model

[0111] The main objective of this study was to improve the inflammatory, oxidative, and aging environment associated with IVDD and promote the recovery of the extracellular matrix within NPCs. To determine the therapeutic effect of HPTC hydrogel on IVDD, we carefully established an in vivo tail needle puncture model of IVDD in rats (…). Figure 6 A). After establishing an IVDD model using a 21G needle puncture, HA-PBA, HPT, and... were administered using a microinjector.

[0112] HPTC hydrogel and PBS (IVDD) were injected into the intervertebral disc (IVD) of rats. Changes in the intervertebral disc (IVD) height index (DHI) and signal intensity were rigorously assessed using X-ray evaluation and magnetic resonance imaging (MRI) results, respectively. Qualitative observation using X-ray ( Figure 6 B) In the HPTC group, the decrease in DHI was not significant, and the images were similar to those in the control group. In the IVDD group, significant IVD intervertebral space collapse and narrowing of the intervertebral space were observed. The HA-PBA and HPT groups were similar to the control group, but still showed slow progression, indicating that the effects of using HA-PBA and HPT hydrogels alone were poor. Figure 6 C). T2-weighted images on magnetic resonance imaging (MRI) are commonly used to characterize the extent of IVDD. As degeneration progresses and significant proteoglycan loss leads to reduced water retention in the IVD, the tissue becomes dehydrated and severely degenerated, appearing as a "black disc" on T2-weighted images, while healthy and well-hydrated tissue appears as a "white disc." MRI images are classified into grades I through V based on signal intensity according to the Pfirrmann classification. MRI images ( Figure 6D) shows healthy intervertebral discs in the control group, with similar signals observed after 8 weeks of HPTC hydrogel treatment. In contrast, the IVDD group showed disc signal loss and interosseous collapse and fusion. In the HA-PBA group, disc signal remained, and degeneration continued, indicating that it has an anti-IVDD effect, but not a sustained or long-lasting one. The HPTC group showed disc signal loss but no bone fusion, suggesting that HPT hydrogel alone can prevent collapse to some extent, but cannot prevent disc signal loss caused by inflammatory necrosis of nucleus pulposus cells.

[0113] Consistent changes in DHI% and MRI scores collectively support the hypothesis of in vivo therapeutic efficacy of HPTC hydrogel. Figure 6 e).

[0114] Histopathological analysis is widely considered a definitive method for diagnosing diseases. To rigorously assess the time-dynamics of treatment efficacy, we performed histological analyses on two different IVD tissues post-surgery. These assessments were systematically performed at 4 and 8 weeks post-treatment application, allowing for longitudinal observation of the treatment's effects on the target tissue over time. H&E staining was the chosen technique to characterize the morphology, fibrous tissue, and margins of the NPs. Results showed that from week 4 to week 8, the NP tissue area in the HPTC hydrogel-treated group showed shrinkage, which was relatively negligible compared to other experimental groups, maintaining clear tissue boundaries. In the IVDD, HA-PBA, and HPT treatment groups, the NPs exhibited progressive degeneration, indistinct margins, and increasing replacement of the central region by annular fibrous tissue. Figure 6 F). Proteoglycans (red) and collagen (blue) in IVD were assessed using safranin-o / fast green staining. (See attached image.) Figure 6 As shown in G, the staining results of the HPTC group and the control group were very similar.

[0115] The IVD center is NP, which is rich in proteoglycans, while other groups (IVDD, HA-PBA, HPT) are gradually replaced by collagen (blue part).

[0116] The therapeutic mechanism of HPTC hydrogel in rat IVDD model

[0117] To fully elucidate the potential of HPTC hydrogel for in vivo applications, in vivo studies were conducted at 4 weeks (Figure S3) and 8 weeks (Figure S4). Figure 7 In step A), the effects of immunohistochemistry on the expression of aggregates, type II collagen, IL-1β, and TNF-α in IVD tissues were assessed. Compared with other experimental cohorts, the HPTC group showed significantly reduced IL-1β and TNF-α signal intensity. Conversely, aggregates and type II collagen (key indicators of ECM content) showed significantly strong signals in the HPTC group. These differences were further confirmed by semi-quantitative staining using IHC scoring. Figure 7 BE).

[0118] Furthermore, the effects of HPTC hydrogel on the hypoxic niche and cellular senescence of the intervertebral disc were critically evaluated. Immunohistochemical analysis showed that HIF-1α expression was significantly increased in the HPTC treatment group compared to the defect group. Figure 7 F, G). Given that HIF-1α is a major regulator coordinating cellular adaptation to hypoxia, its robust enhancement in the HPTC group strongly suggests the successful establishment of a therapeutically beneficial hypoxic microenvironment within NP cells. This regenerative hypoxia, facilitated by the ROS scavenging properties of the hydrogel and reducing excessive oxidative stress, is crucial for maintaining NP cell phenotype and ECM synthesis. Simultaneously, staining for the aging-related marker p21 showed a significant attenuation in the HPTC group compared to the deficient group. Figure 7 The significant reduction in p21 signaling indicates that degenerative processes-induced cellular senescence is effectively alleviated. Overall, the upregulation of HIF-1α and downregulation of p21 highlight the hydrogel's dual capacity to utilize regenerative hypoxia responses and combat age-driven degeneration, thereby targeting fundamental pathological mechanisms beyond inflammation and ECM dysregulation.

[0119] Histological analysis further demonstrated that HPTC-treated intervertebral discs retained NP tissue structure, with reduced inflammation and robust ECM regeneration. These findings collectively indicate that HPTC hydrogel coordinates a three-pronged therapeutic mechanism: restoring hypoxic homeostasis through HIF-1α upregulation, mitigating senescence through p21 downregulation, and rebalancing inflammation and ECM synthesis. While the adherent HA-PBA network enhanced cerium retention to maintain these effects, the multifactorial nature of IVDD warrants further investigation into the long-term bioactivity of HPTC.

[0120] RNA sequencing revealed the potential signaling pathways for HPTC hydrogel treatment of IVDD in rats.

[0121] To further confirm the mechanism of ECM regeneration after HPTC hydrogel treatment, we performed mRNA-seq analysis on NP tissue. Figure 8 A is a volcano plot, showing the differential gene expression profile after HPTC hydrogel treatment. Differentially expressed genes (DEGs) were identified using stringent criteria, particularly statistical significance levels of fold change ≥2 and p < 0.05. Heatmap analysis revealed that these related genes were mainly concentrated in areas related to inflammation, ECM, cell signaling, and cell adhesion. Specifically, the expression of ECM synthesis-related genes Col6a1, Col6a2, and BMP-6, and the hypoxia-related gene Hif-1α was upregulated. Figure 8BC). Gene Ontology (GO) analysis further indicated that these DEGs are mainly involved in ECM activity related to the aforementioned effects of HTPC hydrogel in regulating ECM catabolism / anabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that HPTC hydrogel treatment upregulated the expression of the PI3K-Akt signaling pathway. Figure 8 D). As a key intracellular signaling pathway, the PI3K-Akt signaling pathway has been emphasized in many recent studies for its role in promoting ECM regeneration after activation. The promotion of ECM regeneration in NPCs by HPTC hydrogel may be attributed to the activation of the PI3K-Akt signaling pathway. Gene set enrichment analysis (GSEA) results showed upregulation of Hif-1α, suggesting that HPTC may promote ECM remodeling by reducing ROS and creating a hypoxic environment. Figure 8 E). To confirm the RNA-seq sequencing results, we performed qPCR analysis on Aggrecan, Collagen II, IL-1β, TNF-α, HIF-1α, and p21, which are key biomarkers of inflammatory, oxidative, and aging environments. Figure 8 The results showed that HPTC treatment activated ECM synthesis and downregulated the PI3K / Akt signaling pathway. Simultaneously, upregulation of HIF-1α indicated activation of the cellular adaptive response to hypoxia, while downregulation of p21 indicated blockage of the cellular senescence program.

[0122] The unique cerium-gallate complex in HPTC hydrogel removes excess ROS, thereby coordinating a therapeutically beneficial hypoxic microenvironment, combating age-driven degeneration, and synergistically working with anti-inflammation to achieve overall IVD regeneration.

[0123] In vivo therapeutic effects of HPTC hydrogel on rabbit IVDD model

[0124] The regenerative efficacy of HPTC hydrogel was further validated in a rabbit IVDD model, whose spinal anatomy is closer to the size and biomechanical load of human intervertebral discs. Figure 9 A). Longitudinal radiographic tracking showed different treatment trajectories in each group ( Figure 9 BC). Although the defective group showed progressive intervertebral disc collapse within 8 weeks, HA-PBA and HPT hydrogels showed a moderate but unaffected protective effect against height loss, with early benefits gradually diminishing towards the endpoint. In stark contrast, from week 4 to week 8, HPTC treatment maintained near-physiological intervertebral disc height, similar to the control group. T2-weight MRI confirmed this preservation trend. Figure 9In DE (decalcification), the HPTC-treated intervertebral discs retained high signal intensity similar to healthy, hydrated tissue, while the HA-PBA and HPT groups showed progressive signal attenuation, indicating degenerative progression. Crucially, the histology of undecalcified ground sections provided unprecedented resolution of calcified endplates and the intervertebral disc-bone interface. Histological analysis (…) Figure 9 FG showed that while HA-PBA and HPT moderately delayed nucleus pulposus fibrosis, they failed to prevent endplate erosion and annular tissue destruction. HPTC's uniquely preserved biphasic microstructure—with proteoglycan-rich NP domains (saffron O+) seamlessly integrated with lamellar annular fibrosis and intact cartilaginous endplates—achieved structural fidelity indistinguishable from the control group. This comprehensive regeneration, undetectable in rodent sections, confirms HPTC's ability to restore functional spinal units beyond symptom relief.

[0125] This invention utilizes the dynamic borate ester bond formed between HA-PBA and TA, and the TA-ce bond... 3 A double-crosslinked injectable hydrogel (HPTC) was developed using coordination with +mpn. HPTC hydrogels exhibit excellent rheological properties, injectability, self-healing ability, and tissue adhesion. In vitro experiments showed that HPTC hydrogels alleviated the core pathological features of IVDD by scavenging ROS, reducing oxygen levels, modulating the inflammatory cytokine spectrum, and restoring the HIF-1α signaling pathway. Animal studies in rat and rabbit IVDD models confirmed these findings: intervertebral disc height was maintained, MRI signal intensity was restored, IL-1β and TNF-α levels were reduced, and collagen and aggregate protein expression was increased. Furthermore, RNA-seq analysis showed that HPTC treatment upregulated HIF-1α pathway-related genes, further supporting its regenerative potential. In summary, HPTC hydrogels combine the structural advantages of injectable hydrogels with the therapeutic advantages of metallopolyphenol chemistry, providing a novel minimally invasive strategy for intervertebral disc regeneration, with the potential to restore disc homeostasis and delay or reverse the progression of IVDD.

[0126] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. 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. A method for preparing a cerium metal phenolic network hydrogel, characterized in that, Includes the following steps: Hyaluronic acid-phenylboronic acid, tannic acid and Ce 3+ Solutions with concentrations of 15-20 mg / mL, 30-40 mg / mL, and 15-20 mg / mL were prepared separately, mixed, and then 1-1.2 M NaOH was added to initiate gelation to obtain a cerium metal phenolic network hydrogel; the hyaluronic acid-phenylboronic acid, tannic acid, and Ce... 3+ The molar ratio of NaOH to NaOH is 500-600:100-150:25-30:

1.

2. The method for preparing cerium metal phenolic network hydrogel according to claim 1, characterized in that, The preparation of hyaluronic acid-phenylboronic acid includes the following steps: Hyaluronic acid was dissolved in ultrapure water, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and n-hydroxysuccinimide were added; after stirring, 3-aminophenylboronic acid was added; after the reaction was completed, the product was purified by dialyzing and lyophilized; the molar ratio of hyaluronic acid, -COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, n-hydroxysuccinimide and 3-aminophenylboronic acid was 8-10:1:4-5:2-3:8-10.

3. A cerium metal phenolic network hydrogel, characterized in that, The cerium metal phenolic network hydrogel is prepared by the method described in claim 1 or 2.

4. A method for testing the performance of a cerium metal phenolic network hydrogel, characterized in that, include: Characterization, self-healing, injectability and adhesiveness, dissolved oxygen measurement, in vitro free radical scavenging assay, cytotoxicity detection, hemolysis assay, in vitro antibacterial assay, intracellular ROS scavenging and anti-inflammatory properties, aging-related β-galactosidase staining and RT-qPCR, in vivo evaluation in rat IVDD model and in vivo evaluation in New Zealand white rabbit IVDD model.

5. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The characterization of cerium metal phenolic network hydrogels specifically includes the following steps: adding hyaluronic acid-phenylboronic acid-tannic acid, hyaluronic acid-phenylboronic acid-Ce... + Tannic acid-Ce + A cerium-metal phenolic network hydrogel was dropped onto a glass slide and dried at 60°C; Fourier transform infrared spectra were recorded on a Spectrum 3 spectrometer from 4000 to 400 cm⁻¹. 1 64 scans, resolution: 1cm 1 ; The self-healing, injectability, and adhesion testing specifically included the following steps: Two hydrogel samples were used to observe self-healing, one stained pink with Rhodamine B; each hydrogel was cut in half and reconnected by placing the two halves of different colors at the contact point for 20-30 minutes; a cerium metal phenolic network hydrogel was loaded into a syringe and squeezed into PBS through a 24G needle to test injectability; the cerium metal phenolic network hydrogel was applied to a fractured, isolated rat spine and allowed to adhere for 5-10 minutes to assess adhesion, with the standard of adhesion being no detachment upon standing; all processes were recorded using a digital camera. Dissolved oxygen measurement specifically includes the following steps: Place hydrogen peroxide solution in PBS (pH 6.0-6.5), add Ce... 3+ The reaction takes 16-20 minutes, during which the hydrogen peroxide and Ce react. 3+ The molar ratio was 1:3-4; dissolved oxygen was measured at multiple time points (0, 0.5, 1, 2, 4, 8, 12 and 16 minutes) using a portable oxygen meter.

6. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The in vitro free radical scavenging assay includes the following steps: Exudate from HPTC hydrogel is prepared under pH 6.0-7.5 conditions with a concentration range of 2.5-40 mg / mL. HPTC hydrogel is mixed with PBS at a ratio of 5-10 ml / g, soaked at 37°C for 24-36 h, and the supernatant is collected and filtered sterilized. This supernatant is used to evaluate the scavenging ability of ABTS+·, DPPH·, and ·O2. ABTS and K2S2O8 are reacted in the dark at a molar ratio of 6-7:4-5 for 12-14 h to generate ABTS. + ·; The obtained ABTS + · Solution (OD) 734 =0.70±0.05) and concentrations of 2.5-20 mg / mL -1 The hydrogel secretions were mixed at a volume ratio of 1:1-1.5 and incubated in the dark at 23-25℃ for 30-35 min; the absorbance at 734 nm was recorded on a microplate reader; the ABTS+ clearance rate was calculated using the following formula: In the formula, A0 is the absorbance of ABTS+·reference, A1 is the absorbance of the sample, and A2 is the absorbance of the corresponding solvent reference. DPPH· removal experiment: Add hydrogel exudate to 1-1.2 mL of 0.15-0.2 mM DPPH·ethanol and incubate in the dark at 25°C for 30-35 min; use DPPH· instead of ABTS· as a control, measure absorbance at 517 nm and calculate removal efficiency; • Os2-cavenging experiment: The reaction mixture containing Tris-HCl buffer, pyrogallol, and hydrogel secretion was incubated in the dark at 25°C for 5-10 min; the molar ratio of Tris-HCl, pyrogallol, and hydrogel secretion was 28-30:4-5:6-7; the absorbance values ​​at 325 nm were recorded at 0 min and 5 min; • The Oscavenging rate was calculated using the following formula: Where ΔA0 is the absorbance change of the pyrogallol control, ΔA s It is the change in the absorbance of the sample.

7. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The cytotoxicity assay included the following steps: After incubating HUVECs, 3T3-L1 cells, and NPCs, the culture medium was replaced with an equal volume of fresh culture medium containing hydrogel exudate at a concentration of 0 to 200-250 mg / mL; after further incubation, the culture medium was replaced with one containing calcein AM1-1.2 μg / mL; the fluorescence intensity at 520 nm (excitation: 490 nm) was measured using a microplate reader; cell viability was determined by comparing the fluorescence intensity of the treated samples with that of the untreated control group. The hemolysis test specifically includes the following steps: Prepare a 10-12% red blood cell suspension using fresh rabbit blood; add HA-PBA, TA, and Ce to this suspension. 3+ Alternatively, place the samples in centrifuge tubes containing HPTC hydrogels; test tubes containing deionized water and PBS serve as positive and negative controls, respectively; after incubating at 37°C for 1-1.5 hours, centrifuge the samples at 3300-3500 rpm for 5-7 minutes; record the absorbance of the supernatant at 540 nm using an ELISA reader. Calculate the hemolysis rate using the following formula: The in vitro antibacterial test includes the following steps: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii are selected, and the antibacterial activity of the hydrogel is evaluated using the plate count method; HA-PBA solution or HPTC hydrogel is placed in a centrifuge tube containing 2-2.5 mL of bacterial suspension; after incubation for 6-8 hours, the OD is recorded using a microplate reader. 600 The values ​​were used to quantify bacterial survival on agar plates. The intracellular ROS scavenging and anti-inflammatory properties assays included the following steps: RAW264.7 macrophages were cultured and stimulated with LPS; the control group received only culture medium; LPS-induced cells were incubated with HA-PBA, HPT, or HPTC hydrogels; cells were loaded with DCFH-DA and fluorescence was detected; ROS localization was visualized using CLSM; the expression of IL-1β, IL-4, IL-6, IL-10, and TNF-α was analyzed using RT-qPCR; total RNA was isolated using TRIzol, reverse transcribed, and amplified using SYBR Green premix; GAPDH was used as an endogenous control.

8. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The senescence-related β-galactosidase staining and RT-qPCR assay includes the following steps: NP cells are cultured and incubated with 100-120 μM H2O2 to induce senescence; the control group receives fresh culture medium; the cells are then treated with HA-PBA, HPT, or HPTC hydrogels; senescence is assessed by β-galactosidase staining. The expression of p16, p21, ERK2, HIF-1α and GLUT1 was quantified by RT-qPCR, with GAPDH as an internal control.

9. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The in vivo evaluation of the rat IVDD model included the following steps: Male Sprague-Dawley rats were acclimatized under standard conditions for one week; five animals in each group were randomly divided into: control group (no puncture), PBS group (puncture, injection of 5 μL PBS), HA-PBA group (puncture, injection of 5 μL HA-PBA solution), HPT group (puncture, injection of 5 μL HPT mixed solution), and HPTC hydrogel group (puncture, injection of 5 μL HPTC hydrogel); under 3% isoflurane anesthesia, a 21g puncture needle (5mm depth) was used to percutaneously puncture the coccygeal intervertebral disc (Co3 / 4~Co7 / 8), rotating 360° for 30-60 seconds; immediately after needle removal, 10-15 μL of the corresponding preparation was injected into the nucleus pulposus using a 33-g microsyringe; postoperatively, buprenorphine 0.05mg / kg SC was administered for analgesia for 36-48 hours; Lumbar vertebral discs (IVDs) were examined using a digital X-ray imaging system (Kubtec Model XPERT.8, USA). Subsequently, the disc height index (DHI) was measured. Magnetic resonance imaging (MRI) was performed, and T2-weighted MR images were acquired to assess the water content and structural health of the IVDs. Imaging parameters included a repetition time of 3500 ms, an echo time of 120 ms, a scan range of 200 × 200 mm, and a slice thickness of 1.4 mm to ensure detailed cross-sectional images. Quantitative analysis of the MRI scans included measuring the grayscale values ​​of NPs in different samples using ImageJ software. Intervertebral disc specimens were obtained 4 or 8 weeks later, decalcified in 10% EDTA solution for 1.5-2 months, embedded in paraffin, and then cut into 5 μm thick sections for hematoxylin-eosin (He), saffron O-fast green (SO) staining, and immunohistochemical staining. The sections were imaged using a fluorescence microscope (ECLIPSE Ts2R, Nikon), and the intensity of immunohistochemical staining was semi-quantitatively analyzed using ImageJ software. NP tissues from PBS and HPTC hydrogels were lysed with Trizol reagent and rapidly frozen in liquid nitrogen to stabilize RNA; RNA was extracted and its purity was determined. Chain-specific libraries were prepared and sequenced; raw reads were trimmed, aligned with a reference genome using HISAT2, and counted; expression levels were expressed as the number of fragments per million sequenced fragments per thousand bases of transcripts; differentially expressed genes were identified as DESeq2 (Q<0.05,|log2fold-change|≥1); DEGs were functionally enriched and visualized in R (v3.2.0).

10. The performance testing method for cerium metal phenolic network hydrogel according to claim 4, characterized in that, The in vivo evaluation of the New Zealand white rabbit IVDD model included the following steps: Male New Zealand white rabbits were anesthetized with ketamine / thiazide, and a minimally invasive paramedian approach was used to expose the L1-L6 lumbar vertebrae; an 18-gauge needle was inserted to induce partial rupture of the annulus fibrosus and remove the nucleus pulposus; the groups were divided into: control group (L1-2, no puncture), PBS group (L2-3, puncture and injection of 20-30 μL PBS), HA-PBA group (L3-4, puncture and injection of 20-30 μL HA-PBA solution), HPT group (L4-5, puncture and injection of 20-30 μL HPT mixed solution), and HPTC hydrogel group (L5-6, puncture and injection of 20-30 μL HPTC hydrogel); all injections were completed immediately after puncture; postoperatively, buprenorphine 0.03 mg / kg SC was administered for analgesia for 48-72 hours; X-ray and MRI examinations were performed using the same method as for rat modeling. Intervertebral disc specimens were collected at 4 and 8 weeks of age for grinding and histological examination. The intervertebral discs were cut into 1-2 mm thick slices and completely fixed with neutral formaldehyde buffer. The slices were ground until the disc-like structure was clearly visible. The bone slices were transferred to sufficient water until the washing solution became clear, and then H&E and F&O staining were performed.

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