Antioxidant Proline Carbon Dot‐Composited Poly(L‐methionine) Hydrogel Restores Mitophagy to Alleviate Cellular Senescence in Intervertebral Disc Degeneration

ABSTRACT

The senescence of nucleus pulposus cells (NPCs) is a hallmark pathological feature of intervertebral disc degeneration. Senescent NPCs exhibit mitochondrial dysfunction and impaired mitophagy, thus compromising mitochondrial turnover and quality control. Current therapeutic strategies predominantly target external risk factors but do not restore mitophagy. In this study, a proline carbon dot‐composited poly(L‐methionine) (EG₄₅M₂₅/Pro‐CD) hydrogel is developed and employed to re‐establish the impaired mitophagy pathway. Expression levels of Parkin and phosphorylated Parkin increase, indicating successful activation of the mitophagy pathway. Restoration of mitochondrial function markedly reduces oxidative stress and the senescence‐associated secretory phenotype in NPCs. The levels of type II collagen and aggrecan recover to 84.63% and 77.31% of their normal ones, respectively. In an animal model, the administration of Pro‐CD in a thermo‐sensitive and injectable hydrogel effectively preserves the water content and height of intervertebral discs while maintaining the structural integrity and functional activity of NPCs. The mean Pfirrmann grade improves from 5.00 in the Control group to 3.33 after treatment, whereas the disc height index recovers to 82.06% of the physiological value. Collectively, our findings demonstrate that the EG₄₅M₂₅/Pro‐CD composite hydrogel has therapeutic potential to promote endogenous disc regeneration by enhancing mitophagy, re‐establishing redox homeostasis, and alleviating NPC senescence.

Antioxidant proline carbon dot‐composited poly(L‐methionine) hydrogel restores impaired mitophagy in senescent nucleus pulposus cells by enhancing Parkin activation, improving mitochondrial function to reduce oxidative stress and the senescence‐associated secretory phenotype and preserving disc hydration, height, and structural integrity in vivo, thereby re‐establishing redox homeostasis and promoting endogenous regeneration in intervertebral disc degeneration.

1. Introduction

Intervertebral disc degeneration (IVDD) is a prevalent degenerative spinal disorder and a principal pathological basis for numerous spine‐related diseases, constituting a major cause of lower back pain [1]. Epidemiological data indicate that IVDD accounts for approximately 40% of all cases of lower back pain and affects almost 600 million individuals worldwide, substantially impairing their quality of life [2]. Currently prevalent clinical management primarily relies on conservative or surgical interventions, which are largely palliative and focus on symptomatic relief rather than the correction of underlying disc pathology [3]. Therefore, enhancing the endogenous repair of intervertebral discs has emerged as an important therapeutic strategy for IVDD.

Nucleus pulposus cells (NPCs) are the key functional units of the intervertebral disc [4]. The aberrant senescence and death of NPCs are known to disrupt metabolic homeostasis in the extracellular matrices (ECMs), ultimately causing irreversible structural damage to the intervertebral disc, thus representing a central pathogenic driver of IVDD [5]. Current biomaterial‐based therapeutic strategies are directed almost exclusively to the extracellular microenvironments and either mitigate deleterious cues or supplement functional components of the ECMs [6]. Consequently, the reversal of NPC senescence is a critical prerequisite for activating endogenous repair processes in IVDD [7]. The dysfunction of mitophagy, a crucial upstream driver of cellular senescence, initiates a pathological cascade in NPCs [8]. Moreover, the inefficient clearance of damaged mitochondria results in mitochondrial functional disorders, leading to abnormal metabolism, redox imbalance, and cell‐cycle arrest, collectively exacerbating the senescent phenotype [9]. Previous studies have demonstrated that the restoration of mitophagy in senescent cells alleviated the senescent phenotype and restored physiological functionality [10]. Therefore, targeting mitophagy to mitigate NPC senescence represents a promising endogenous therapeutic strategy for IVDD.

In this study, a proline carbon dot‐composited poly(L‐methionine) (EG₄₅M₂₅/Pro‐CD) hydrogel was developed to rescue impaired mitophagy (Scheme 1). Proline (Pro), a non‐essential and ubiquitous amino acid in animals, inherently exhibits excellent biocompatibility and water solubility. Furthermore, unlike linear amino acids, the unique pyrrolidine ring structure of Pro predisposes it to form a defined carbon (C) core upon carbonization. As a precursor for collagen synthesis, Pro has been demonstrated to regulate mitophagy and mitigate cellular senescence [11]. Consequently, Pro acted as a C source and had significant potential to confer additional biological functions. Through carbonization and nanoscale structuring, Pro‐CD exhibited a markedly enlarged surface area and enhanced cellular uptake. The poly(ethylene glycol)₄₅−poly(ε‐methacryloyl‐L‐methionine) (EG₄₅M₂₅) hydrogel possessed thermo‐sensitive and reactive oxygen species (ROS)‐scavenging properties, facilitated sustained in situ release of Pro‐CD, and improved the microenvironments in IVDD, serving as an optimal carrier for Pro‐CD. Experimental analysis demonstrated that this composite system significantly attenuated the progression of IVDD in a rat model. Collectively, these findings identify Pro‐CD encapsulated in EG₄₅M₂₅ as a promising nanotherapeutic platform that reduces oxidative damage, alleviates cellular senescence, and restores mitophagy in IVDD.

SCHEME 1.

Pro‐CD composite hydrogel alleviates oxidative damage and cellular senescence by promoting mitophagy, thereby rescuing mitochondrial function.

2. Results and Discussion

2.1. Oxidative Stress and Mitochondrial Dysfunction as Drivers of IVDD

To elucidate pathological mechanisms associated with IVDD, the GSE147383 dataset from the Gene Expression Omnibus (GEO) database was firstly analyzed (Figure 1A). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed prominent alterations in oxidative stress, cell‐cycle processes, lipid metabolism, and ECM remodeling (Figure 1B−D). The interplay between these dysregulated mechanisms outlines a complex pathological network and highlights the central contribution of NPC dysfunction to the progression of IVDD. Oxidative stress serves as an upstream driver by inducing the excessive production of ROS, which directly injures NPCs [12]. This oxidative insult precipitates both senescence and apoptosis, diminishes the NPC population, and impairs intrinsic reparative capacity. Within the pathological microenvironments of IVDD, an excess of ROS drives cellular senescence via multiple molecular pathways. First, ROS directly induces DNA damage, leading to cell cycle arrest. An excess of ROS will further amplify the senescence‐associated secretory phenotype (SASP), promoting the expression of matrix‐degrading enzymes and inflammatory factors, thereby forming a degenerative vicious cycle [7]. In addition, sustained oxidative stress leads to a reduction in mitochondrial membrane potential, impaired functionality of the electron transport chain, and mitochondrial DNA (mtDNA) damage. This compromises cellular adenosine triphosphate (ATP) synthesis and triggers aberrant mitophagy and biosynthetic imbalances. Consequently, damaged mitochondria release additional ROS and proapoptotic factors, thus creating a self‐amplifying loop of cellular injury [13]. Ultimately, this cumulative imbalance leads to the loss of critical structural components and progressive disc collapse.

FIGURE 1.

Analysis of human NP samples in GEO Database. (A) Volcano plot of differential gene expression of normal and degenerative NP tissues. (B) GO analysis of significantly differentially expressed genes. (C) KEGG analysis of significantly differentially expressed genes. (D) Heatmap of gene expression associated with both GO and KEGG enrichment analysis.

Pathway analysis also revealed considerable enrichment for terms, such as “mitochondrial depolarization” and “mitochondrial outer membrane”, indicating pronounced mitochondrial impairment in NPCs. These findings emphasized the pivotal role of mitochondrial dysfunction in driving oxidative stress, senescence, and metabolic imbalance [14]. Mitophagy is a key regulatory pathway that maintains mitochondrial turnover and removes damaged organelles, thereby maintaining redox homeostasis and metabolic stability [15]. However, oxidative stress and cellular senescence exert marked suppression on mitophagy [16]. Therefore, enhancing mitophagy to alleviate oxidative stress and suppress NPC senescence represents a promising therapeutic strategy.

2.2. Synthesis and Characterizations of Pro‐CD and EG₄₅M₂₅ Hydrogel

Carbon dots (CDs), an emerging class of zero‐dimensional nanomaterials, have attracted substantial interest due to their excellent cellular permeability and high biocompatibility [17]. In this study, Pro‐CD was synthesized through a one‐step thermal process using Pro as the sole precursor (Figure 2A). The nanoscale dimensions of Pro‐CD facilitated efficient cellular internalization and enabled diverse biological applications. Transmission electron microscopy (TEM) confirmed that Pro‐CD exhibited uniform spherical morphology with a mean diameter of 2.17 ± 0.16 nm. The lattice spacing of 0.32 nm corresponded to the (002) plane of graphitic C, thus verifying the successful formation of crystalline CDs from Pro precursors (Figure 2B,C) [18].

FIGURE 2.

Physical and chemical structures of Pro‐CD. (A) Synthesis pathway of Pro‐CD. (B) Representative TEM image of Pro‐CD. (C) Particle size distributions of Pro‐CD. (D) FT‐IR spectra of Pro and Pro‐CD. (E) XRD pattern of Pro and Pro‐CD. (F) Full‐scan XPS survey spectra for Pro‐CD. (G−I) High‐resolution C 1s, O 1s, and N 1s XPS spectrum for Pro‐CD. (J) Photoluminescence spectra of Pro‐CD.

FIGURE 10.

In vivo histological evaluation of therapeutic efficacy of EG₄₅M₂₅/Pro‐CD for IVDD. (A) Represent H&E and S&FG staining of Co7/8 segments of rat caudal vertebrae. (B) Heatmap of histological grades across treatment groups at 1, 4, and 8 weeks after operation. (C) Represent Acan and Col II immunohistochemical staining of Co7/8 segments of rat caudal vertebrae. (D) Histological scores for each treatment group. All statistical data are represented as mean ± SD (n = 3; **p < 0.01, ***p < 0.001).

To elucidate functional group transformations and characterize structural evolution, Fourier transform‐infrared (FT‐IR) spectroscopy was performed on both Pro and the resulting Pro‐CD (Figure 2D). The FT‐IR spectrum exhibited a broad absorption band at 3450 cm⁻¹, attributable to overlapping O─H and N─H stretching vibrations, thus indicating that the hydrophilic surface groups and nitrogen (N)‐containing moieties from the Pro precursor were retained in Pro‐CD. Absorption peaks at 2800−2900 cm⁻¹ correspond to C−H stretching of aliphatic hydrocarbons, thus reflecting the saturated C framework of pyrrolidine ring. In Pro, a distinct C─O stretching band was evident at 1650 cm⁻¹. This is a characteristic feature of free carboxyl groups, and this peak disappeared after carbonization, thus suggesting that C─O group underwent condensation with available N─H or O─H bonds. Concurrently, new absorption bands emerged at 1630−1660 and 1500−1550 cm⁻¹, assigned to amide I and amide II vibrations, respectively. These features reflected contributions from newly generated C─O bonds in amide and ester linkages and from C─C stretching in conjugated aromatic domains formed during carbonization. The peaks at 1510 and 1330 cm⁻¹ correspond to N─H bending and C−N stretching vibrations, respectively.x X‐ray diffraction (XRD) analysis revealed a broad peak at 20°−30° (2θ), characteristic of the (002) plane of graphitic C (Figure 2E), thus confirming the presence of graphitic domains along with functionalized surfaces in Pro‐CD.

X‐ray photoelectron spectroscopy (XPS) was used to resolve further the chemical states and bonding configurations of Pro‐CD (Figure 2F). The material consisted of 73.07 at% C, 12.36 at% N, and 14.57 at% oxygen (O), closely matching the theoretical stoichiometry of Pro and indicating effective N retention during carbonization. Deconvolution of the C 1s spectrum identified four characteristic components, consistent with the partial retention of carboxyl functionalities (Figure 2G). The O 1s spectrum revealed peaks corresponding to O−C─O groups, indicating that a subset underwent dehydrogenation to form ether linkages (Figure 2H). The N 1s spectrum exhibited two dominant species: A major pyrrolidine‐type secondary amine (−NH−, 399.6 eV) reflecting the preservation of the Pro ring structure and a minor protonated amine (−NH₂ ⁺, 401.2 eV), suggesting the partial participation of N─H in condensation reactions forming the emerging conjugated network (Figure 2I).

Excitation‐dependent photoluminescence, a hallmark of CD, was observed in Pro‐CD. Increasing excitation wavelengths from 340 to 480 nm produced a progressive redshift in emission from 440 to 540 nm (Figure 2J). Pro‐CD exhibited its strongest excitation at 420 nm with a corresponding emission maximum at 512 nm. Ultraviolet−visible (UV−Vis) spectra revealed characteristic absorption bands between 290 and 320 nm (Figure S1), attributable to n−π* transitions of C─O groups and π−π* transitions of conjugated C─C structures, consistent with the optical signatures of C‐based nanomaterials.

EG₄₅M₂₅ was synthesized by ring‐opening polymerization (ROP) of L‐methionine N‐carboxyanhydride (Met NCA) using amino‐terminated methoxy polyethylene glycol (mPEG‐NH₂, number‐average molecular weight (M _n) = 2000 g mol⁻¹) as a macroinitiator [19]. Successful polymerization was confirmed by ¹H nuclear magnetic resonance (NMR) spectrum (Figure 3A). Quantitative ¹H NMR analysis indicated an average degree of polymerization of Met unit of 25 based on integration of the Met methine proton (−C(O)CH(CH₂−)NH−, 4.8 ppm) relative to terminal mPEG methylene signals (−CH ₂CH ₂O−, 3.8 ppm). FT‐IR spectra further exhibited characteristic bands at 3300 cm⁻¹ (N─H stretching of secondary amines), 1650 cm⁻¹ (C─O stretching), 1270 cm⁻¹ (C─O─C stretching), and 1100 cm⁻¹ (C─S─C stretching), collectively confirming the successful synthesis of EG₄₅M₂₅ (Figure 3B).

FIGURE 3.

Physical and chemical structures of EG₄₅M₂₅. (A) ¹H NMR spectrum of EG₄₅M₂₅. (B) FT‐IR spectra of EG₄₅M₂₅. (C) Phase transition diagram of sol‐to‐gel transition of EG₄₅M₂₅. (D) Temperature‐dependent rheological properties of EG₄₅M₂₅. (E) Representative TEM image of EG₄₅M₂₅. (F) Pore size distributions of EG₄₅M₂₅. (G) Temperature‐dependent changes in circular dichroism spectrum of EG₄₅M₂₅. (H) Quantitative analysis of temperature‐dependent changes in circular dichroism spectrum. (I) ¹H NMR spectrum of EG₄₅M₂₅ solution oxidized by H₂O₂ in deuterium oxide (D₂O). (J) Quantitative analysis of ¹H NMR spectra of EG₄₅M₂₅ solution oxidized by H₂O₂. (K) Circular dichroism spectra of EG₄₅M₂₅ solution oxidized by H₂O_2. (L) Quantitative analysis of circular dichroism spectra of EG₄₅M₂₅ solution oxidized by H₂O_2. (M) Mass loss profiles of EG₄₅M₂₅ hydrogel in PBS with varying H₂O₂ concentrations. (N) ABTS radical scavenging rate of different concentrations of EG₄₅M₂₅. (O) DPPH radical scavenging rate of different concentrations of EG₄₅M₂₅. All statistical data are represented as mean ± SD (n = 3).

The physicochemical properties of EG₄₅M₂₅ hydrogel were systematically evaluated. Analysis revealed that EG₄₅M₂₅ exhibited pronounced thermo‐sensitivity, with phase‐transition analysis revealing an inverse relationship between polymer concentration and gelation temperature. As concentration increased from 5.0 to 9.0 wt.%, the gelation temperature decreased from 31.0 to 18.0°C (Figure 3C). Considering the relatively low body temperature of intervertebral discs in the rat model, and the need for injectability, a 6.0 wt.% concentration was used in subsequent experiments. Mechanical characterizations revealed sharp increases in both the storage modulus (G′) and the loss modulus (G″) near the gelation point, indicating a rapid transformation from a liquid‐like sol to an elastic gel network (Figure 3D). At 20°C, G′ measured 2 Pa, rising to 299 Pa at 35°C. The low G′ prior to gelation enabled efficient drug incorporation and supports minimally invasive injection with reduced tissue damage. Cryo‐scanning electron microscopy (SEM) imaging further confirmed an interconnected porous microarchitecture with a mean pore diameter of 4.05 ± 0.57 µm and a porosity exceeding 50%, and these features are structurally suitable for cellular attachment and tissue regeneration (Figure 3E,F).

Next, circular dichroism spectroscopy was used to probe the temperature‐dependent secondary structural transitions underlying the thermo‐sensitive behavior of EG₄₅M₂₅ (Figure 3G). Sol−gel transition was primarily driven by PEG‐block dehydration, coupled with conformational rearrangements in the poly(amino acid) segments, which collectively promoted micelle aggregation and gelation. As the temperature increased from 10 to 60°C, the α‐helix content decreased from 32.59% to 23.55%, whereas the β‐sheet content increased from 10.59% to 15.64%, and random coil content remained essentially unchanged (Figure 3H). Dehydration of PEG, together with conformational changes in EG₄₅M₂₅, induced micellar aggregation, driving the sol–gel transition.

Sulfur ether groups endowed EG₄₅M₂₅ with intrinsic ROS‐scavenging reactivity, producing hydrophilic sulfoxide and sulfone groups upon oxidation. To investigate this mechanism, hydrogen peroxide (H₂O₂), the most abundant form of physiological ROS, was adopted as a probe. ¹H NMR spectroscopy revealed the progressive emergence and intensification of resonance signals at 4.4−4.3, 3.0−2.5, and 2.3−1.9 ppm during the reaction (Figure 3I). This indicates that EG₄₅M₂₅ participates in the reaction, leading to the formation of hydrophilic sulfoxide and sulfone groups. Quantitative analysis revealed reaction completion within five days at 40.0 mmol L⁻¹ H₂O₂, with lower concentrations reducing efficiency and prolonging the reaction time (Figure 3J). Circular dichroism spectroscopy further revealed that exposure to ROS increased α‐helix content (28.19% to 58.22%) and decreased β‐sheet content (12.66% to 4.89%), a pattern that contrasted with that observed during temperature‐induced gelation. These results suggested that the exposure to ROS shifted EG₄₅M₂₅ toward a sol‐like secondary structural state (Figure 3K,L). The changes in secondary structure further indicated that oxidation of Met‐containing segments disrupted the original chain conformation, thereby attenuating phase separation. Degradation assays performed at varying H₂O₂ concentrations revealed a concentration‐dependent degradation profile (Figure 3M). After 16 days, the hydrogel retained more than 30% of its mass in phosphate‐buffered saline (PBS) but only 13% in 500.0 µM H₂O₂, thus confirming its capacity to neutralize excessive ROS.

Based on this ROS‐reactive behavior, antioxidant performance was assessed by performing 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assays. EG₄₅M₂₅ exhibited concentration‐dependent scavenging activity, reaching 22.05% (ABTS) and 24.15% (DPPH) at 2000 µg mL⁻¹ (Figure 3N,O). Biocompatibility testing further demonstrated high cell viability (>90%) and minimal hemolysis (<0.5%), consistent with previously reported cytocompatibility and antioxidant efficacy (Figure S2) [20]. This cytoprotective effect was further corroborated by live/dead staining (Figure S3).

Collectively, the temperature‐triggered gelation of EG₄₅M₂₅ minimized implantation‐induced tissue trauma and prevented Pro‐CD leakage, whereas its high water content and ROS‐scavenging capability established a supportive microenvironment.

2.3. Pro‐CD‐Preserved Viability and Functionality of NPCs in vitro

Excellent biocompatibility is essential if Pro‐CD is to be utilized biologically. NPCs exhibited >90% viability even at 4000 µg mL⁻¹, thus confirming the favorable biosafety of Pro‐CD (Figure S4). Cellular uptake studies using intrinsic fluorescence revealed efficient internalization, with predominant cytoplasmic localization. Fluorescence intensity increased over the first 9 h, and subsequently declined, thus suggesting efficient intracellular processing and metabolism (Figure 4A,B). Following the generation of CDs, Pro‐CD formed a nanoplatform with a high density of surface functional groups, achieved through molecular cross‐linking and rearrangement. The nanoscale size will facilitate cellular entry via endocytosis and enhance cellular retention. Furthermore, the carbonized cores of Pro‐CD neutralized various forms of ROS more effectively than Pro monomers, thereby regulating cellular redox homeostasis more efficiently (Figure S5). This is attributed to the sp^{2 −}hybridized C core of CDs and the resulting π–π conjugation, which enhances the material's electron‐donating capacity. Previous studies reported that CDs derived solely from selenomethionine exhibited 3.1‐fold higher antioxidant capacity than their precursors [21].

FIGURE 4.

Protective effects of Pro‐CD against oxidative damage. (A) Representative fluorescence microscopy images showing cellular uptake of Pro‐CD by NPCs. (B) Quantitative analysis of integrated intensity Pro‐CD. (C) Representative fluorescence microscopy images of ROS levels after co‐incubation with H₂O₂, Pro, or Pro‐CD. (D) Quantitative analysis of fluorescent staining images of C. (E) Representative fluorescence microscopy images of 8‐OHdG after co‐incubation with H₂O₂, Pro, or Pro‐CD. (F) Quantitative analysis of fluorescent staining images of E. (G) Flow cytometry of anti‐apoptotic effect after co‐incubation with H₂O₂, Pro, or Pro‐CD. (H) Statistical analysis of total apoptosis rate from flow cytometry data shown in G. (I) Representative fluorescence microscopy images of cleaved caspase‐3 and iNOS after co‐incubation with H₂O₂, Pro, or Pro‐CD. (J, K) Quantitative analysis of fluorescent staining images of I. (L) WB of cleaved caspase‐3 contents. (M) Semi‐quantitative analysis of cleaved caspase‐3 levels. All statistical data are represented as mean ± SD (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Within the pathological landscape of IVDD, excessive ROS‐driven senescence and the aberrant apoptosis of NPCs constitute a central pathogenic axis. This detrimental microenvironments, together with cellular dysfunction, reinforce a self‐amplifying cycle that accelerates degeneration [12]. To assess the protective effects of Pro‐CD, an oxidative injury model was established in NPCs using H₂O₂. Cell Counting Kit‐8 (CCK‐8) assays revealed a reduction in the viability of NPCs to 59.58% of the Normal group following H₂O₂ exposure. In contrast, cotreatment with Pro‐CD preserved cell viability, with the most substantial effect observed at 200 µg mL⁻¹, where viability remained 85.53% of that in the Normal group (Figure S6). Furthermore, treatment with 6.0 wt.% of EG₄₅M₂₅ in combination with various concentrations of Pro‐CD consistently yielded a coefficient of drug interaction (CDI) < 1, indicating a synergistic effect and enhanced efficacy in alleviating oxidative damage (Tables S1 and S2). The levels of intracellular ROS were visualized by a DCFH‐DA fluorescent probe (Figure 4C). Compared with the oxidative damage control group, Pro treatment reduced ROS level by 40.94%, whereas Pro‐CD produced a more pronounced reduction of 76.71% (Figure 4D; Figure S7). Dihydroethidium staining also revealed a significant reduction in superoxide anion level (Figure S8). Oxidative DNA damage was further assessed by detecting 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a key biomarker of oxidative DNA injury [22]. Immunofluorescence analysis confirmed that Pro‐CD provided superior protection, reducing 8‐OHdG increase level by 70.50%, a substantially greater reduction than the 38.23% observed with Pro alone (Figure 4E,F).

Excessive levels of oxidative stress trigger apoptosis in NPCs, aggravating metabolic dysfunction within the nucleus pulposus (NP) and accelerating the progression of IVDD. To determine whether Pro‐CD mitigates aberrant apoptosis, Annexin V‐FITC/PI flow cytometry was performed (Figure 4G). Pro‐CD significantly attenuated H₂O₂‐induced apoptosis, reducing the total apoptosis rate from 41.34% to 16.95%, whereas treatment with the Pro monomer only reduced apoptosis to 32.39% (Figure 4H). Given its role as a central executor of apoptosis, cleaved caspase‐3 expression was quantified by immunofluorescence and Western blot (WB) analysis (Figure 4I,L). Results of both assays demonstrated a marked suppression of cleaved caspase‐3 by Pro‐CD, with expression reducing to 72.61% and 40.23% of control level, respectively (Figure 4J,M; Figure S9). Furthermore, immunofluorescence results revealed that Pro‐CD significantly downregulated inducible nitric oxide synthase (iNOS), a proapoptotic mediator, by 14.63% relative to the oxidative damage control group (Figure 4I,K) [23]. Collectively, these results indicate that Pro‐CD effectively counteracted apoptosis induced by oxidative stress and preserved the viability of NPCs under oxidative stress.

NPCs are the principal cells governing ECM metabolism within the NP, and their senescence disrupts metabolic homeostasis [5]. Immunofluorescence staining of γH2A.X revealed that Pro‐CD reduced DNA double‐strand breaks to 17.19% of control level (Figure 5A,B). Similarly, SA‐β‐gal staining revealed a marked reduction in the proportion of senescent cells following Pro‐CD treatment (Figure S10). Senescent NPCs adopt a SASP phenotype that skews ECM metabolism toward catabolism, characterized by elevated expression of degradative enzymes and reduced synthesis of anabolic components such as type II collagen (Col II) and aggrecan (Acan) [24]. Matrix metalloproteinase‐13 (MMP‐13), a key driver of ECM degradation and a hallmark of IVDD, was markedly suppressed by Pro‐CD, with fluorescence intensities reduced by 47.42% compared with the oxidative control group (Figure 5A,C). Furthermore, the fluorescence intensities of Col II and Acan in the Pro‐CD group recovered to 90.88% and 96.16% of normal levels, respectively (Figure 5D,E).

FIGURE 5.

Anti‐aging properties of Pro‐CD. (A) Representative fluorescence microscopy images of γH2A.X, MMP‐13, Col II, and Acan after co‐incubation with H₂O₂, Pro, or Pro‐CD. (B−E) Quantitative analysis of fluorescent staining images of A. (F) WB of SASP contents, including Col II, Acan, ADAMTS‐5, MMP‐3, MMP‐13, IL‐6, p16, and p21. (G−N) Semi‐quantitative analysis of SASP levels. All statistical data are represented as mean ± SD (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

These findings were corroborated by WB. Under oxidative stress, the expression levels of Col II and Acan decreased to 26.03% and 34.40% of normal ones, respectively. Pro‐CD treatment substantially reversed these declines, restoring Col II and Acan to 84.63% and 77.31% of normal levels (Figure 5F−H). In addition, Pro‐CD significantly reduced the expression levels of key catabolic mediators of ECM degradation. Relative to the oxidative damage group, the protein levels of a disintegrin and metalloproteinase with thrombospondin motifs‐5 (ADAMTS‐5), MMP‐3, MMP‐13, and interleukin‐6 (IL‐6) were reduced by 47.96%, 51.42%, 73.48%, and 31.02%, respectively (Figure 5F,I−L; Figures S11−S16).

Another hallmark of cellular senescence is the dysregulation of cell‐cycle control accompanied by persistent DNA double‐strand breaks [25]. p16 and p21 are widely recognized as negative regulators of the cell cycle and are commonly used as markers of cellular senescence. WB analysis showed that oxidative stress markedly increased the expression of p16 and p21 by 3.92‐ and 2.41‐fold, respectively, relative to the Normal group (Figure 5F,M,N; Figures S17 and S18). Pro‐CD treatment substantially mitigated this senescence‐associated arrest, reducing p16 and p21 levels to 1.95‐ and 1.66‐fold of normal, respectively. Consistent with these findings, quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis of multiple SASP markers, including ADAMTS‐5, MMP‐3, MMP‐13, IL‐6, p16, and p21, revealed significant transcriptional downregulation following Pro‐CD treatment, further supporting its antisenescence activity at the gene‐expression level (Figure S19). To further validate activation of the IL‐6‐mediated JAK–STAT3 signaling pathway, key signaling mediators involved in ECM degradation—such as phosphorylated JAK and STAT3—were significantly downregulated. In contrast, the expression of Sox9, a transcription factor that promotes matrix synthesis, was markedly upregulated (Figures S20 and S21). Together, these data demonstrate that Pro‐CD effectively attenuated cellular senescence and preserved the normal secretory phenotype of NPCs, thereby maintaining metabolic homeostasis in the ECMs.

In conclusion, Pro‐CD was efficiently internalized by NPCs to alleviate oxidative stress‐induced injury, including ROS accumulation, apoptosis, and senescence. By exerting these protective effects, Pro‐CD sustained the physiological functionality of NPCs and supported metabolic balance in the ECMs.

2.4. Identification of Therapeutic Mechanism Underlying Ability of Pro‐CD to Attenuate Senescence in NPCs

Mitochondria are central regulators of cellular energy metabolism and redox homeostasis and are also major sources of ROS. Previous evidence indicated that Pro enhances mitophagy, thereby promoting mitochondrial homeostasis [11]. MitoTracker staining and TOM20 immunofluorescence were used to evaluate mitochondrial status (Figure 6A). Oxidative stress induced severe mitochondrial dysfunction, reducing MitoTracker and TOM20 fluorescence to 18.46% and 31.93% of normal levels. Pro‐CD markedly reversed these changes, restoring fluorescence to 58.43% and 80.39% of physiological values, respectively. Because elevated mitochondrial superoxide (O₂·⁻) reflects oxidative mitochondrial injury, MitoSOX staining was performed [26]. Oxidative stress increased mitochondrial O₂·⁻ to 4.66‐fold of the normal level, whereas Pro‐CD led to a reduction of 1.51‐fold (Figure 6C−E). The use of JC‐1 staining to assess mitochondrial membrane potential (ΔΨ_m) confirmed the pronounced loss of ΔΨ_m under oxidative stress, with the aggregate‐to‐monomer ratio falling to 4.45% of normal. Pro‐CD treatment effectively restored ΔΨm, increasing the ratio to 56.52% of normal (Figure 6B,F). To further assess mitochondrial function, Seahorse analysis was performed to measure oxygen (O₂) consumption rate (OCR) under various conditions. Compared to the oxidative damage group, NPCs treated with Pro‐CD exhibited recovery in basal respiration, ATP production, maximal respiration, and spare respiratory capacity (Figure S22). These results indicate restoration of both mitochondrial function and cellular energy metabolism.

FIGURE 6.

Promoting mitophagy properties of Pro‐CD. (A) Representative fluorescence microscopy images of Mitotracker, MitoSox, and TOM 20 after co‐incubation with H₂O₂, Pro, or Pro‐CD. (B) Representative fluorescence microscopy images of JC‐1 staining after co‐incubation with H₂O₂, Pro, or Pro‐CD. (C−E) Quantitative analysis of fluorescent staining images of A. (F) Quantitative analysis of fluorescent staining images of B. (G) Representative fluorescence microscopy images of Parkin, LC3B, and p62 after co‐incubation with H₂O₂, Pro, ord Pro‐CD. (H) Representative TEM images of NPC after co‐incubation with H₂O₂ and Pro‐CD. (I) Representative WB images of LC3B, Parkin, p‐Parkin, and p62 contents. (J) Semi‐quantitative analysis of LC3B, Parkin, p‐Parkin, and p62 levels. All statistical data are represented as mean ± SD (n = 3; *p < 0.05, **p < 0.01, ***P< 0.001, ****p < 0.0001).

Given reports that Pro enhance mitophagy by upregulating Parkin, mitophagy‐associated markers were further examined (Figure 6G) [11]. Immunofluorescence analysis revealed that oxidative stress significantly elevated Parkin and LC3B expression to 1.29‐ and 5.27‐fold, respectively, above normal ones, suggesting partial mitophagy activation, while p62 accumulation increased to 1.75‐fold, indicating impaired mitophagy flux. Pro‐CD significantly improved mitophagy: Parkin and LC3B increased to 2.02‐ and 21.03‐fold of normal ones, and p62 declined to 1.29‐fold (Figure S23). WB analysis corroborated these findings, further showing that Pro‐CD elevated phosphorylated Parkin (p‐Parkin) level to 2.24‐fold of that in normal cells, thus confirming activation of Parkin‐dependent mitophagy (Figure 6I,J; Figures S24−S27). qRT‐PCR analysis further revealed the transcriptional changes in these genes (Figure S28).

Ultrastructural abnormalities often accompany mitochondrial dysfunction [27]. TEM imaging revealed pronounced mitophagy in Pro‐CD‐treated cells (Figure 6H). Quantitative morphometry demonstrated that oxidative stress reduced mitochondrial cross‐sectional area and aspect ratio (AR) by 88.23% and 49.70%, respectively. However, Pro‐CD treatment restored mitochondrial area to 61.11% and AR to 82.06% of normal values (Figure S29). These results further support the notion that Pro‐CD promoted the clearance of damaged mitochondria and restored normal mitochondrial morphology by enhancing mitophagy.

To further investigate whether Pro‐CD was able to reverse metabolic dysfunction and cellular senescence via the regulation of mitophagy, rather than solely through alleviation of oxidative stress. Mdivi‐1 and Urolithin A were used as inhibitors and activators, respectively. WB analysis revealed that Mdivi‐1 reduced Col II protein expression and increased p16 protein expression, thereby counteracting the therapeutic effect of Pro‐CD. Conversely, Urolithin A reversed this trend, an effect consistent with that observed following Pro‐CD treatment (Figures S30 and S31). Collectively, these results indicated that Pro‐CD preserved mitochondrial homeostasis by enhancing mitophagy, thereby counteracting oxidative stress‐induced IVDD characterized by cellular senescence and metabolic dysfunction.

Given the pronounced protective effects of Pro‐CD on NPCs in vitro, the mechanisms underlying these effects were further interrogated using integrated metabolomic and transcriptomic analysis. KEGG enrichment of the metabolomic dataset revealed that Pro‐CD markedly modulated key biological processes in NPCs, including amino acid metabolism, oxidative stress responses, and energy homeostasis, thus indicating broad and multifaceted metabolic reprogramming (Figure 7A,B). At the metabolite level, treatment with Pro‐CD increased redox‐regulating molecules, such as glutathione and pyridoxal, and was accompanied by a concomitant decline in oxidative damage markers, including uric acid and nitrotyrosine (Figure 7C) [28, 29, 30]. This normalization of redox coincided with metabolic signatures indicative of mitochondrial function restoration, reflected in the recovery of lactic acid, pyruvic acid, and creatine levels [31, 32]. Moreover, metabolites essential for ECM biosynthesis, including hydroxyproline and mannose were significantly elevated [33, 34]. Cluster analysis of differentially regulated metabolites further demonstrated reduced oxidative stress and restored energy and substrate metabolism in NPCs (Figure 7D). Collectively, these metabolomic data suggest that Pro‐CD rescued mitochondrial dysfunction under oxidative stress, thereby enhancing the anabolic capacity required for ECM synthesis.

FIGURE 7.

Cellular metabolomics analysis of NPC. (A) Volcano plot of differential metabolite abundance of control and Pro‐CD‐treated NPC. (B) KEGG analysis of significantly different metabolites. (C) Heatmap of differential metabolites across different treatment groups. (D) Clustering analysis of significantly different metabolites across different treatment groups.

Transcriptome sequencing was then performed to elucidate the molecular pathways by which Pro‐CD exerted its regulatory effects. Pro‐CD induced a broad transcriptional response in NPCs (Figure 8A). GO enrichment analysis revealed significant modulation of biological processes central to tissue remodeling, ECM metabolism, cell‐cycle regulation, and mitophagy, consistent with the in vitro observations (Figure 8B,C). KEGG pathway analysis further confirmed that Pro‐CD modulated genes associated with oxidative stress, cell‐cycle progression, ECM metabolism, and intercellular interactions (Figure 8D,E). Generation of a corresponding heatmap illustrated the robust reversal of aberrant gene expression patterns following Pro‐CD treatment (Figure 8F).

FIGURE 8.

Cellular transcriptomics analysis of NPC. (A) Volcano plot of differential gene expression between control and Pro‐CD‐treated NPC. (B) GO analysis of significantly differentially expressed genes between control and Pro‐CD‐treated NPC. (C) Chord diagram of interactions between GO terms and associated genes. (D) KEGG analysis of significantly differentially expressed genes between control and Pro‐CD‐treated NPC. (E) Chord diagram of interactions between KEGG pathway and associated genes. (F) Heatmap of gene expression associated with both GO and KEGG enrichment analysis.

In conclusion, integrated metabolomic and transcriptomic profiling revealed that Pro‐CD exerted therapeutic effects predominantly by enhancing mitophagy, thereby attenuating oxidative stress and cellular senescence.

2.5. Pro‐CD‐Composited EG₂₅M₂₅ Hydrogel Mitigated IVDD in vivo

Building on the observed anti‐senescent and promitophagy activities of Pro‐CD in oxidative stress‐induced NPCs in vitro, an EG₄₅M₂₅/Pro‐CD composite hydrogel was developed and evaluated for the effects of this hydrogel in a rat tail puncture model of IVDD. The study design and experimental timeline are shown in Figure 9A. Magnetic resonance imaging (MRI) and digital radiography (DR) and magnetic resonance imaging (MRI) were performed at 1‐, 4‐, and 8‐weeks post‐surgery, and intervertebral disc tissue was collected at 4‐ and 8‐weeks for histological analysis.

FIGURE 9.

In vivo imaging evaluation of therapeutic efficacy of EG₄₅M₂₅/Pro‐CD for IVDD. (A) Schematic representation of IVDD model and treatment protocol. (B) Represent MRI images of Co7/8 segments of rat caudal vertebrae at 1, 4, and 8 weeks after operation. (C) Heatmap of Pfirrmann grades across treatment groups at 1, 4, and 8 weeks after operation. (D) Represent DR images of Co7/8 segments of rat caudal vertebrae at 1, 4, and 8 weeks after operation. (E) Heatmap of DHI as a percentage across treatment groups at 1, 4, and 8 weeks after operation. All statistical data are represented as mean ± SD (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The in vivo safety of composite hydrogel system is a prerequisite for its potential application. Histological analysis of the major organs confirmed the favorable biocompatibility of this system (Figure S32). As shown in Figure 9B, the MRI signal intensity in the saline group was markedly reduced compared with the Normal group and continued to diminish over time, indicating the progressive loss of disc hydration. The EG₄₅M₂₅ hydrogel alone partially preserved disc hydration, attributable to its intrinsic water‐retentive and antioxidant properties. Notably, the incorporation of Pro‐CD into the EG₄₅M₂₅ hydrogel further mitigated the decline in water content. Collectively, these findings demonstrated that the combined EG₄₅M₂₅/Pro‐CD system effectively slowed the progression of IVDD in vivo. Quantification using the Pfirrmann grading system revealed that the positive control group exhibited mean grades of 4.00, 4.33, and 5.00 at 1‐, 4‐, and 8‐weeks, respectively. In contrast, animals treated with the Pro‐CD‐loaded hydrogel exhibited lower grades of 3.33, 3.00, and 3.33 at the same time points, further confirming the therapeutic benefit (Figure 9C; Figure S33A).

Disc height index (DHI), a key indicator of disc structural integrity, was quantified over time from X‐ray images (Figure 9D). In the saline group, DHI declined to 79.97% of physiological values at one week and declined further to 67.14% by eight weeks, thus reflecting ongoing degenerative collapse. In comparison, the EG₄₅M₂₅/Pro‐CD composite hydrogel preserved 96.33% of baseline disc height at one week and 82.06% at eight weeks, thus demonstrating a substantial protective effect against disc height loss (Figure 9E; Figure S33B). Together, these results indicate that the composite hydrogel effectively preserved intervertebral disc structure and functionality in vivo.

To further characterize microstructural alterations within the intervertebral discs, hematoxylin and eosin (H&E) and safranin O−fast green (S&FG) staining were used to assess histological changes in the segments undergoing surgery (Figure 10A; Figure S34). In the Normal group, disc tissue exhibited a well‐organized architecture, with clearly defined NP boundaries and intact NP morphology. In contrast, the saline group exhibited progressive structural collapse: The nucleus pulposu−sannulus fibrosus (NP−AF) interface had become indistinct, and by eight weeks, NP tissue was almost undetectable. The EG₄₅M₂₅ group retained partial NP structure at one week, but by 4 and 8 weeks, the NP tissue was no longer clearly identifiable, indicating that antioxidant activity alone was insufficient to provide sustained protection against IVDD. In striking contrast, the EG₄₅M₂₅/Pro‐CD composite hydrogel preserved well‐defined NP morphology and distinct annular boundaries even at eight weeks. These findings demonstrate that the composite hydrogel maintained native disc microarchitecture and mitigated degenerative progression. Quantitative histological scoring further supported these observations. The saline group showed progressive deterioration, with mean scores of 10.33, 11.00, and 11.67 at 1‐, 4‐, and 8‐weeks, respectively. Conversely, the EG₄₅M₂₅/Pro‐CD hydrogel achieved improved outcomes, with scores of 5.00, 5.33, and 5.67 at the corresponding time points. Together, these results confirm the ability of composite hydrogel to attenuate IVDD effectively (Figure 10B,D).

These protective effects were further corroborated by immunohistochemical staining of Col II and Acan in the NP region (Figure 10C). Normal discs exhibited robust staining for both markers, consistent with high ECM content in healthy NP tissue. The saline group exhibited almost complete loss of NP tissue and minimal expression of Col II and Acan, consistent with advanced IVDD. The EG₄₅M₂₅ group retained partial ECM expression at four weeks but failed to prevent subsequent matrix degradation, underscoring the limited therapeutic efficacy of EG₄₅M₂₅ hydrogel monotherapy. Notably, the EG₄₅M₂₅/Pro‐CD composite hydrogel enhanced Col II and Acan expression, demonstrating its ability to promote ECM remodeling and restore metabolic homeostasis in NP tissue.

Taken together, imaging and histological analysis demonstrated that the EG₄₅M₂₅/Pro‐CD composite hydrogel provided superior protection against IVDD compared to the hydrogel alone. This combination treatment maintained disc hydration, preserved disc height, re‐established matrix homeostasis, and enhanced metabolic balance. These findings are consistent with in vitro observations supporting Pro‐CD as a promising agent for mitigating NPC senescence and slowing disc degeneration. Building on the in vivo findings, further optimization of the formulation composition is expected to support further the clinical translational potential of this composite hydrogel system.

3. Conclusion

In this study, a thermo‐sensitive EG₄₅M₂₅/Pro‐CD composite hydrogel was synthesized, allowing for injectable in situ gelation within a disc and forming a microenvironment conducive to the survival of NPCs. Pro‐CD was synthesized from a single Pro monomer and retained the functional groups of the precursor while exhibiting a characteristic graphite‐like C structure. In vitro, Pro‐CD effectively restored redox balance under oxidative stress, scavenging approximately 80% of excess ROS and reducing apoptosis by 24.39%. This intervention attenuated the SASP and re‐established metabolic homeostasis in the ECMs, with Col II and Acan levels returning to 84.63% and 77.31% of their physiological ones, respectively. Simultaneously, the expression levels of catabolic factors, including MMP‐3, MMP‐13, ADAMTS‐5, and IL‐6, were markedly suppressed.

Mechanistically, Pro‐CD restored impaired mitophagy flux, thereby facilitating the clearance of damaged mitochondria. This restoration was supported by a 67.55% reduction in mitochondrial superoxide (O₂·⁻), the recovery of mitochondrial membrane potential (ΔΨm) and morphology, and a 26.66% reduction in accumulated p62 when compared with the oxidative damage group. Concurrently, Parkin and p‐Parkin expression increased by 2.36‐ and 2.24‐fold, respectively. In subsequent in vivo studies, imaging analysis further confirmed the effective preservation of water content in NP and DHI. By eight weeks post‐surgery, EG₄₅M₂₅/Pro‐CD composite hydrogel maintained a DHI of 82.06% and improved the Pfirrmann grade from 5.00 to 3.33 in the degeneration model. These macroscopic improvements aligned with histological evidence of preserved disc microarchitecture and essential NP biochemical components. Histological examination specifically demonstrated the robust retention of key NP ECM constituents. The present study still has some limitations. While the EG₄₅M₂₅/Pro‐CD composite hydrogel demonstrated promising therapeutic effects, its mechanical properties have not yet been fully optimized to mimic the native tissue. Therefore, future iterations of the hydrogel will be formulated better to match the mechanical properties of the native tissue. Such modifications are intended to provide a stiffness range that closely recapitulates the native NP mechanical microenvironments. Collectively, these findings establish a promising nanomaterial‐based therapeutic strategy for IVDD. This approach shows substantial clinical potential by reversing cellular senescence to promote endogenous repair.

4. Experimental Section

This section describes the synthesis of Pro‐CD and EG₄₅M₂₅ hydrogel, the isolation of NPCs, the surgical methods used to generate an animal model of IVDD, and the statistical methods used for data analysis. Other methods and materials are provided in Supporting Information.

4.1. Preparation of Pro‐CD and EG₄₅M₂₅ Hydrogel

Pro‐CD was synthesized via one‐step pyrolysis using a Pro precursor. The precursor was ground, heated at 240°C, and the resulting product was dispersed in water. After sonication, the suspension was centrifuged and filtered to remove aggregates. The filtrate was further purified by dialysis (molecular weight cut off (MWCO) = 500 Da) for 24 h, followed by freeze‐drying to obtain the final Pro‐CDs.

In a dry three‐necked flask equipped with a nitrogen (N₂) inlet, L‐Met was suspended in anhydrous tetrahydrofuran. Triphosgene was added to the suspension, and the mixture was stirred at 60°C for 60 min under a N₂ atmosphere. The formation of a clear solution indicated complete consumption of the starting material. After purging the system with N₂ for 30 min, the resulting solution was concentrated to approximately 30.0 mL under reduced pressure. The concentrate was precipitated into 300.0 mL of ice‐cold n‐hexane. The upper layer was decanted, affording the crude product as a light yellow liquid. The crude NCA intermediate was subsequently dissolved in 150.0 mL of chilled ethyl acetate, and the organic phase was washed three times with ice‐cold water. Anhydrous magnesium sulfate was added to remove residual water, and the mixture was stored at −20°C overnight to allow further drying. The desiccant was removed by filtration through a G4 sintered glass funnel. The purified Met NCA was obtained by evaporating the ethyl acetate under reduced pressure.

EG₄₅M₂₅ was synthesized by the ROP of Met NCA using mPEG‐NH₂ as the macroinitiator. In brief, mPEG₄₅‐NH₂ was dissolved in anhydrous N,N‐dimethylformamide (DMF) after dehydration by azeotropic distillation. Met NCA was added to the macroinitiator solution, and the polymerization was carried out under a N₂ atmosphere at room temperature for 72 h. The reaction product was purified by precipitation in excess diethyl ether three times. The collected precipitate was redissolved in DMF and dialyzed against deionized water using a dialysis membrane (MWCO 2000 Da) for 72 h. Finally, the purified copolymer EG₄₅M₂₅ was obtained as a white solid after lyophilization.

4.2. Isolation and Culture of Rat NPCs

NPCs were isolated from three 10‐week‐old female Sprague‐Dawley rats that had been euthanized by isoflurane overdose. Carcasses were surface‐sterilized with 75% (V/V) ethanol. Under aseptic conditions in a laminar flow hood, NP tissue was dissected and transferred into DMEM/F12 medium. The tissue was then digested with 0.5% (W/V) type II collagenase for 3 h at 37°C. The digest was passed through a 70 µm cell strainer and centrifuged at 1200 rpm for 5 min. Next, the cell pellet was resuspended in complete DMEM/F12 medium supplemented with 10% (V/V) fetal bovine serum (FBS) and 2% (V/V) penicillin‐streptomycin. NPCs were maintained in a humidified incubator at 37°C with 5% (V/V) carbon dioxide (CO₂).

4.3. Cell Viability Analysis

Cytotoxicity was assessed with the CCK‐8 assay. NPCs were seeded at a density of 7,000 cells per well in 96‐well plates and cultured for 24 h at 37°C under 5% (V/V) CO₂. The cells were then exposed to DMEM/F12 medium containing different concentrations of Pro‐CD, a precursor amino acid, or EG₄₅M₂₅ for 24 h. After treatment, 10% (V/V) CCK‐8 reagent in DMEM/F12 medium was added to each well, and absorbance at 450 nm was recorded with a microplate reader. To examine the protective effects of Pro‐CD and precursor amino acids under oxidative stress, NPCs were plated under identical conditions. After 24 h of incubation, cells were co‐treated with various concentrations of the test compounds and H₂O₂ at a final concentration of 200.0 µM for an additional 24 h. Cell viability was then determined using the CCK‐8 assay as described above.

4.4. Immunofluorescent Staining

After the treatments described above, cells from four groups were fixed with 4.0% (W/V) paraformaldehyde (PFA) for 15 min at room temperature and permeabilized using 0.25% (V/V) Triton X‐100 in PBS for 15 min. After blocking with 5.0% (W/V) bovine serum albumin (BSA) for 1 h at 37°C to reduce non‐specific binding, the cells were incubated overnight at 4°C with the primary antibody against vinculin, prepared in PBS containing 1.0% (W/V) BSA. After incubation, the cells were washed three times with immunostaining wash buffer and incubated for 90 min at room temperature with appropriate fluorescently labeled secondary antibody and DAPI. Imaging was performed using a confocal laser scanning microscopy, and fluorescence intensity was analyzed with ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).

4.5. Animal Model and Surgical Procedure

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Changchun Institute of Applied Chemistry (Approval number: CIAC 2023[0116]) and conducted in accordance with relevant guidelines and regulations. Forty‐eight female Sprague‐Dawley rats (eight weeks of age) were randomly divided into four groups (n = 12 per group): Normal, Control, EG₄₅M₂₅, and EG₄₅M₂₅/Pro‐CD. The Normal group did not receive surgery or any treatment throughout the study, serving as a healthy control.

Following the confirmation of adequate anesthesia, the surgical site was disinfected with povidone iodine. The Co7/8 coccygeal intervertebral disc was located, and a sterile 21‐gauge needle was inserted vertically, rotated 720°, and held in place for 30 s to induce consistent and severe disc injury. With the needle held in place, a micro‐injector was then used to administer the formulations into the disc. The Control group received 10.0 µL of PBS, and the EG₄₅M₂₅ group received 10.0 µL of the hydrogel matrix. The EG₄₅M₂₅/Pro‐CD group received a composite formulation containing Pro‐CD at 200.0 µg mL⁻¹. The injection site was marked on the skin with black ink for subsequent identification. Therapeutic efficacy was assessed at 1, 4, and 8 weeks post‐surgery. Following imaging, the animals were euthanized, and the caudal spinal segments were harvested for further analysis.

4.6. Magnetic Resonance Imaging and Digital Radiography Analysis

MRI was conducted with a T2‐weighted sequence. T2 signal intensity, which reflects the water content of NP, was used to indicate disc hydration and degenerative status. The severity of intervertebral disc degeneration was evaluated using the Pfirrmann grading system, which ranges from Grade I (non‐degenerated) to Grade V (severely degenerated). This system assesses signal intensity, the homogeneity of the NP, the distinction between the NP and the annulus fibrosus, and disc height. Higher Pfirrmann grades correspond to progressively advanced degenerative changes. DR was performed to assess changes in DHI.

4.7. Statistical Analysis

All raw data were first examined for technical errors. Data from replicate experiments (n = 3) were averaged and normalized to the Normal group. All quantitative data are represented as mean ± standard deviation (SD). Statistical analysis was conducted using IBM SPSS Statistics (version 26.0.0; IBM Corp., Armonk, NY, USA). Comparisons between two groups were performed using the Student's t‐test, preceded by a Levene's test for equality of variances. Comparisons between three or more groups were performed using one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test. p > 0.05 was not significant (NS). *p < 0.05 was considered statistically significant. Significance levels are indicated as follows: **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Author Contributions

Z.L. performed conceptualization, validation, formal analysis, investigation, resources, wrote the original draft, and reviewed and edited the final manuscript. Q.Z. performed investigation, resources, and reviewed and edited the final manuscript. H.Y. reviewed and edited the final manuscript. C.F. performed conceptualization, supervision, project administration, acquired funding acquisition, and reviewed and edited the final manuscript. Y.S. performed conceptualization, supervision, project administration, reviewed, and edited the final manuscript. J.D. performed conceptualization, supervision, project administration, acquired funding acquisition, and reviewed and edited the final manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (Grant Nos. U21A2099, 52403394, and 82071391), the Science and Technology Development Program of Jilin Province (Grant No. 20230204089YY, 20250102075JC), and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant No. Y2023066).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: adma72951‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.