Synergistic microenvironment therapy: a dual-function nanogel of supramolecular collagen ll-mimetic matrix and antioxidant MXene for attenuating intervertebral disc degeneration

Abstract

Intervertebral disc degeneration (IVDD) is driven by severe oxidative stress, a loss of extracellular matrix (ECM) homeostasis, and ferroptosis of nucleus pulposus cells (NPCs) as a core pathological mechanism. Therapeutic strategies based on microenvironment engineering that can simultaneously combat oxidative damage and promote tissue repair are urgently needed. Here, we reestablish the IVDD microenvironment using a dual-function nanogel, which integrates a catalytic 2D Mo₂C MXene within a supramolecular Collagen II-mimetic matrix formed by a GFOGER-functionalized self-assembling peptide hydrogel (SAPH). The function of clearing oxidative stress be attribute to the catalytic Mo₂C MXene and the SAPH-R₁₆GFOGER serving as an effective local depot, which to preserve mitochondrial integrity and dismantle the ferroptotic program. Concurrently, the SAPH-R₁₆GFOGER mimic Collagen II matrix to promoting NPC adhesion and proliferation, upregulating the synthesis of new ECM, and shifting the cellular phenotype from catabolic to anabolic. It is notable that in a rat IVDD model, this dual-function engineering strategy effectively mitigated oxidative stress, preserved disc height and hydration, and restored the native ECM-rich architecture of the nucleus pulposus. This work presents a synergistic microenvironment engineering strategy, demonstrating that the integration of an antioxidant MXene within a biomimetic matrix is a promising platform for attenuating degenerative diseases.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04211-w.

Introduction

Low Back Pain represents a major global public health challenge, with its profound impact on disability often underestimated compared to its effect on mortality [1]. As the single leading cause of disability worldwide, Low Back Pain affected an estimated 619 million people in 2020—a figure projected to rise to 843 million by 2050 due to population growth and aging [2]. This staggering prevalence creates an immense burden on public health systems and socioeconomic development, a reality further highlighted by national studies showing a notable persistence of disability despite available treatments [3]. Among the various etiologies driving this crisis, Intervertebral Disc Degeneration (IVDD) is a primary pathological basis for chronic, discogenic pain [4]. The core pathological feature of IVDD is the progressive degradation of the extracellular matrix (ECM), which leads to a loss of disc height, impaired hydration, biomechanical dysfunction [5], and a sharp decline in the endogenous cell population of the nucleus pulposus (NP) [6]. This process is exacerbated by a complex degenerative microenvironment characterized by oxidative stress, chronic low-grade inflammation, aberrant mechanical stress, and pathological neurovascular ingrowth [7–9]. Consequently, the pathogenesis of IVDD is a multifactorial process involving the intricate interplay and dysregulation of multiple molecular pathways, including cellular senescence, apoptosis, impaired autophagy, and, as recently identified, ferroptosis [10, 11].

Ferroptosis is a unique form of regulated cell death driven by iron-dependent lipid peroxidation, characterized by the catastrophic accumulation of lipid peroxides and dysregulation of intracellular iron homeostasis [12, 13]. First systematically described and named in a seminal 2012 study, it was established as a distinct cell death modality, with unique morphological and biochemical features that differentiate it from apoptosis and other forms of programmed cell death [14]. Its molecular mechanism centrally involves iron overload and the functional failure of the key antioxidant hub—the GSH/glutathione peroxidase 4 (GPX4) axis [15–17]. Ferroptosis is now recognized as a central and particularly destructive driver of the degenerative cascade in IVDD [10, 11]. This rapid form of iron-dependent cell death initiates a vicious cycle of lipid peroxidation and catastrophic oxidative stress, which rapidly accelerates the loss of nucleus pulposus cells and the breakdown of the extracellular matrix. Therefore, the ability to specifically neutralize ferroptosis has emerged as a key challenge that must be overcome for effective therapeutic intervention. Intriguingly, a complex regulatory crosstalk exists between ferroptosis and apoptosis. Excessive reactive oxygen species (ROS) can both trigger lipid peroxidation to drive ferroptosis and activate mitochondria-dependent apoptotic pathways [18, 19], with shared upstream regulators like p53 and the mitochondria serving as a critical regulatory node in this network [20–22]. As the primary source of ROS, preserving mitochondrial homeostasis is essential for protecting disc cells from these intertwined death pathways.

Given the central role of oxidative stress in driving both ferroptosis and apoptosis, the development of biomaterials that can precisely scavenge ROS and restore cellular redox balance represents a frontier in regenerative therapies for IVDD. Recently, 2D transition metal carbides/nitrides (MXenes) have emerged as highly promising multifunctional nanomaterials in biomedicine due to their unique physicochemical properties [23]. MXenes such as Ti₃C₂T_X and V₂C exhibit potent, broad-spectrum ROS-scavenging capabilities, attributed to their ability to mimic natural enzymes like superoxide dismutase (SOD) and catalase (CAT); this allows them not only to remove excess ROS but also to efficiently convert superoxide and H₂O₂ into benign oxygen [24, 25]. These properties, alongside excellent biocompatibility and pro-regenerative potential, make them ideal candidates for mitigating oxidative stress in degenerative diseases [26, 27]. Notably, we selected Mo₂C nanosheets for this study, distinguished not only by their potent ROS-scavenging activity but also by the favorable biosafety profile associated with molybdenum, an essential trace element [24]. We thus hypothesized that the powerful antioxidant nature of Mo₂C could be uniquely harnessed to disrupt ferroptosis—a previously unexplored therapeutic strategy for IVDD. However, the safe and effective delivery of these nanoparticles to the degenerative disc, while providing a supportive microenvironment for cell survival and function, remains a critical challenge.

Self-assembling peptide hydrogels (SAPHs) offer an ideal platform to address this challenge, as their nanofibrous architecture closely mimics the natural ECM [28]. These biocompatible and injectable scaffolds provide a 3D biomimetic microenvironment that supports essential cell behaviors, including adhesion, proliferation, and differentiation. Furthermore, supramolecular SAPHs exhibit robust biological functionality, attributable to the incorporation of designed active sequences and the augmentation of functionality achieved by self-assembly. Their bioactivity can be enhanced through precise functionalization to deliver specific biochemical cues. For instance, Yu et al. demonstrated that a RADARADARADARADA (R₁₆) hydrogel delivering TGF-β1 could effectively induce the chondrogenic differentiation of BMSCs [29]. Similarly, Zhao et al. developed a glycopeptide hydrogel where an RGD motif provided cell adhesion sites and a fucoidan component conferred antioxidant function by activating the NRF2 pathway to scavenge ROS and improve the inflammatory microenvironment [30]. The collagen-mimetic peptide GFOGER is another potent functional sequence that specifically binds to integrin receptors crucial for ECM-mediated cell signaling [31, 32]. This interaction activates intracellular signaling cascades, including the FAK-MAPK pathway, to effectively direct cell fate and promote ECM synthesis and remodeling [33, 34].

Herein, we designed and constructed a novel multifunctional nanogel (Mo₂C@SAPH-R₁₆GFOGER) to treat IVDD by synergistically targeting oxidative stress, ferroptosis, and ECM homeostasis (Scheme1). We are convinced that combining the superior ROS-scavenging Mo₂C nanosheets with a GFOGER-functionalized self-assembling biomimetic peptide hydrogel could create a synergistic therapeutic microenvironment. This artificial environment would not only neutralize deleterious ROS to inhibit ferroptosis and apoptosis but also actively promote cell adhesion and ECM synthesis, leading to a more robust and comprehensive restoration of the degenerated disc than either component could achieve alone. This work systematically evaluates the physicochemical properties, biocompatibility, and in vitro capacity of this composite nanogel to protect nucleus pulposus cells from oxidative damage and ferroptosis. In addition, the functional mechanism of composite nanogel materials in promoting ECM regeneration, anti-oxidation and inhibiting ferroptosis was proved through transcriptomics and signal pathway studies. More importantly, we validate its therapeutic potential in vivo using a rat needle-puncture model of IVDD, aiming to provide an innovative, advanced biomaterial-based solution for the clinical treatment of IVDD.

Scheme 1.

Schematic illustration of the synthesis and therapeutic mechanism of the Mo₂C@SAPH-R₁₆GFOGER nanogel for the treatment of intervertebral disc degeneration

Results and discussion

Progressive human intervertebral disc degeneration is characterized by severe extracellular matrix degradation and a pro-ferroptotic molecular signature

To establish the key pathological features of IVDD and identify relevant therapeutic targets, we first performed a comprehensive analysis of human NP tissues of varying degenerative grades (Pfirrmann Grade II-V). As expected, T2-weighted magnetic resonance imaging (MRI) revealed a grade-dependent loss of the bright signal indicative of severe dehydration, along with a progressive narrowing of the disc space which confirms the macroscopic structural failure of the disc unit (Fig. 1A). This radiological assessment correlated directly with a profound deterioration of tissue architecture at the histological level. Alcian Blue and Safranin O/Fast Green staining demonstrated a stark and progressive depletion of the proteoglycan- and glycosaminoglycan-rich matrix, which is essential for the disc’s biomechanical function. In severely degenerated discs (Grade V), this vital matrix was almost entirely replaced by disorganized, fibrous scar-like tissue, and chondrocyte-like cell clusters appeared, indicating a pathological shift in tissue phenotype (Fig. 1B). This change from NP cells to chondrocyte-like clusters is a known adaptative or degenerative response to the altered mechanical and nutritional environment [35]. Quantitative analysis confirmed a significant, grade-dependent decrease in both relative GAG content and Safranin O staining intensity (Fig.S1).

Fig. 1.

Progressive Human Intervertebral Disc Degeneration Is Characterized by Extracellular Matrix Degradation and a Pro-Ferroptotic Molecular Signature. (A) T2-weighted magnetic resonance images (MRI) of human lumbar spines. (B) Corresponding histological staining of nucleus pulposus tissue. Scale bar: 200 μm. Alcian Blue staining (top row), Safranin O/Fast Green (SO&FG) staining (bottom row). (C) Western blot analysis of ACAN, MMP-13, GPX4, and FHC protein expression in human NP tissues of different degenerative grades. GAPDH was used as a loading control. (D) Dot plot visualization of the relative protein expression changes quantified from Fig. 1C. (E) Representative immunohistochemical staining for ACAN, MMP-13, ACSL4, and GPX4 in NP tissues across different grades of degeneration. Scale bar: 200 μm. (F) Semi-quantitative analysis of the immunohistochemical scores for ACAN and GPX4 from Fig. 1E. (G) Semi-quantitative analysis of the immunohistochemical scores for MMP-13 and ACSL4 from Fig. 1E. (H) Schematic diagram summarizing the key pathological changes in a degenerated nucleus pulposus. Data are presented as mean ± SD (n = 5). p < 0.05, ****p < 0.0001; ns, not significant

At the molecular level, this structural breakdown was clearly driven by a distinct shift from an anabolic to a catabolic state. Western blot analysis, visualized both as bands and a corresponding dot plot, confirmed that the expression of the core matrix protein Aggrecan (ACAN) was significantly and progressively downregulated with increasing degenerative grade (Fig. 1C, D). Concurrently, the expression of Matrix Metallopeptidase 13 (MMP-13), a key enzyme responsible for collagen and proteoglycan degradation [36], was markedly upregulated, particularly in Grade IV and V tissues where matrix destruction was most severe (Fig. 1C, D). Beyond this classic view of matrix degradation, we investigated the role of ferroptosis, a form of regulated cell death increasingly implicated in degenerative diseases. Notably, we found a strong correlation between the severity of degeneration and the emergence of a pro-ferroptotic molecular signature. Immunohistochemical staining and Western blot analysis consistently showed that the expression of GPX4, the master negative regulator of ferroptosis that protects against lipid peroxidation, was significantly diminished in degenerated tissues. This loss of primary antioxidant defense was accompanied by a decrease in Ferritin Heavy Chain (FHC), suggesting dysregulation of iron storage and potentially increased levels of a labile intracellular iron pool. Furthermore, we observed a significant upregulation of the pro-ferroptotic enzyme Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) (Fig. 1C-G).This specific combination of downregulated GPX4 and upregulated ACSL4 is now considered a molecular signature of active ferroptosis [37].

The concurrent loss of NP matrix integrity and the robust activation of a pro-ferroptotic state, summarized in the schematic diagram (Fig. 1H), suggest that ferroptosis is not merely a bystander event but is a critical contributing mechanism to the cellular dysfunction and tissue failure that drive IVDD progression. These findings link the structural failure of the disc directly to a pro-ferroptotic molecular shift, thereby substantiating the rationality and effectiveness of our therapeutic strategy of simultaneously targeting ferroptosis and restoring ECM homeostasis.

Synthesis, multiscale characterization, biocompatibility and retention of the injectable Mo₂C@SAPH-R₁₆GFOGER composite hydrogel

To construct a functional and biomimetic nanogel, we first designed a self-assembling peptide, R₁₆GFOGER (R₁₆ is assembly unit [38, 39]; GFOGER is bioactive unit), which were synthesized using the Fmoc-based solid-phase peptide synthesis approach (Fig. 2A). The successful synthesis and purity (> 95%) of this functionalized peptide were rigorously validated by High-performance liquid chromatography (HPLC) and mass spectrometry (MS) (Fig. S2-3). 20 mg R₁₆GFOGER peptide powder is dissolved in aqueous solution, it can self-assemble into supramolecular nanofibers, and then wind into nanogel.

Fig. 2.

Synthesis, Multiscale Characterization, and Biocompatibility of the Injectable Mo₂C@SAPH-R₁₆GFOGER Composite Hydrogel (A) Chemical structure of the functionalized R₁₆GFOGER peptide. (B) TEM images of the nanofibrous network of R₁₆ and R₁₆GFOGER hydrogels. Scale bar: 200 nm. (C) FTIR analysis of R₁₆ and R₁₆GFOGER peptides. (D) Evaluation of the fluorescence spectra of Th-T in R₁₆ and R₁₆GFOGER. (E) Rheological strain sweep test. (F) Rheological cyclic shear recovery test demonstrating the self-healing properties of the hydrogel. (G) SEM images of lyophilized R₁₆ and R₁₆GFOGER hydrogels. Scale bar: 50 μm. (H) Demonstration of the sol-gel transition and injectability of the Mo₂C-loaded hydrogel precursor solution. (I) In vitro degradation profiles of the R₁₆ and R₁₆GFOGER hydrogels over 28 days. (J) TEM image of Mo₂C nanosheets. Scale bar: 20 nm. (K) Bio-TEM image showing the internalization of Mo₂C@SAPH-R₁₆GFOGER nanocomposites by NPCs. Scale bar: 500 nm. (L) Proliferation of NPCs cultured on different hydrogels. (M) In vivo fluorescence imaging showing the retention of free Mo₂C versus Mo₂C@SAPH-R₁₆GFOGER hydrogel in rat tail discs over 3 weeks. Data are presented as mean ± SD. *p < 0.05; ns, not significant

The self-assembled nanogel was characterized at multiple scales. The spectra for both R₁₆ and R₁₆GFOGER showed a characteristic strong absorption peak around 1666 cm⁻¹, which is indicative of the amide I band signals and the β-sheet conformation essential for assembly (Fig. 2B). To confirm the molecular basis for this self-assembly, Fourier-transform infrared spectroscopy (FTIR) was performed. At the nanoscale, transmission electron microscopy (TEM) revealed the base R₁₆ peptide and the completed R₁₆GFOGER peptide all can form a network of fine, uniform nanofibers (Fig. 2C) [40]. Supramolecular nanofiber formation of R₁₆ and R₁₆GFOGER in solution was investigated using a Thioflavin T (ThT) assay. As shown in Fig. 2D, the fluorescence intensities exhibited positive peaks at 480 nm.

Rheological analysis confirmed that this structural remodeling resulted in enhanced mechanical properties. A strain sweep test demonstrated that the R₁₆GFOGER hydrogel was mechanically stiffer and stronger than the base R₁₆ hydrogel, exhibiting a substantially higher storage modulus (G’) (Fig. 2E). Crucially for its potential clinical application, the hydrogel also displayed excellent shear-thinning and rapid self-healing capabilities. This thixotropic behavior is desirable for clinical translation, as it allows for easy injection through a needle followed by rapid in situ resolidification [41]. Its G’ fully recovered within 3 min after the cessation of high strain, confirming its suitability as an injectable material (Fig. 2F). At the microscale, scanning electron microscopy (SEM) of the lyophilized scaffolds demonstrated an aligned, lamellar structure that more closely mimics the native ECM, whereas the base R₁₆ hydrogel exhibited a random, interconnected fibrous network (Fig. 2G).

Macroscopically, the precursor solution containing Mo₂C nanosheets underwent a rapid sol-gel transition within 10 min at 37 °C to form a stable composite nanogel. Its successful formation and excellent injectability were visually confirmed (Fig. 2H). Following formation, the degradation behavior was evaluated in vitro. As shown in Fig. 2I, both R₁₆ and R₁₆GFOGER hydrogels exhibited a sustained degradation profile, losing approximately 90% of their mass over 28 days, which matches the pace of tissue repair.

Next, the therapeutic component, molybdenum carbide (Mo₂C) nanosheets, was characterized. TEM imaging revealed its ultrathin, 2D sheet-like morphology (Fig. 2J). To confirm whether the nanocomposite could be internalized by cells to exert its biological functions, we performed Bio-TEM analysis. As presented in Fig. 2K, electron-dense aggregates corresponding to the Mo₂C nanocomposites were clearly observed within the intracellular vesicles of NPCs, confirming their successful internalization. A cytotoxicity assessment showed that Mo₂C exhibited no significant cytotoxicity towards nucleus pulposus cells (NPCs) at a concentration of 30 µg/mL, with cells maintaining favorable viability. Thereafter, this concentration was applied for all subsequent experiments (Fig. S4).

Finally, the biocompatibility and in vivo retention were assessed. The blood compatibility was verified by a hemolysis assay, indicating its excellent safety profile (Fig. S5). A CCK-8 assay demonstrated that the composite nanogel effectively supported cell proliferation (Fig. 2L). Furthermore, Live/Dead staining (Fig. S6) and a crystal violet cell adhesion assay (Fig. S7) confirmed high cell viability and robust cell attachment. A critical requirement for an effective local therapy is the sustained retention of the therapeutic agent. In vivo fluorescence imaging demonstrated that the SAPH-R16GFOGER hydrogel served as an effective local depot, significantly prolonging the retention of Mo2C nanosheets within the intervertebral disc for over 3 weeks (Fig. 2K). To further evaluate systemic safety for clinical translation, we conducted a 4-week in vivo study. Serum analysis showed that markers for liver (ALT, AST) and kidney function (CRE, BUN), inflammation (IL-1β, IL-6), and oxidative stress (MDA, SOD/GSH) remained at normal levels at all time points (Fig. S8, S9). Moreover, ex vivo fluorescence imaging of major organs (heart, liver, spleen, lung, kidney, brain) harvested at 4 weeks detected no significant signal (Fig. S10), confirming that the material was strictly confined to the injection site without systemic leakage.

In summary, these data demonstrate that the Mo₂C@R₁₆GFOGER nanogel possesses suitable mechanical properties, a porous architecture, injectability, and excellent cytocompatibility, making it a promising candidate for intervertebral disc therapy.

The composite hydrogel substrate fosters a pro-anabolic and anti-catabolic microenvironment for adherent nucleus pulposus cells

To evaluate the capacity of our hydrogel system to function as a pro-regenerative substrate for nucleus pulposus cells (NPCs), we cultured the cells on the surface of pre-formed hydrogels: the basic self-assembling peptide hydrogel (SAPH)-SAPH-R₁₆, the functionalized SAPH-R₁₆GFOGER, and the composite Mo₂C@SAPH-R₁₆GFOGER (Fig. 3A). After a period of culture, we assessed the deposition of a newly synthesized extracellular matrix (ECM) layer using histological staining. As shown by Alcian Blue and Toluidine Blue staining, NPCs cultured on the Mo₂C@SAPH-R₁₆GFOGER substrate produced the denser and more intensely stained matrix layer than other hydrogels, indicating the robust production of NP-specific ECM (Fig. 3B). Quantitative analysis of the integrated optical density (IOD) from these stains corroborated these visual findings, showing a statistically significant, stepwise increase in matrix synthesis from SAPH-R₁₆ to SAPH-R₁₆GFOGER, and peaking on the Mo₂C@SAPH-R₁₆GFOGER substrate (Fig. S11).

Fig. 3.

The Composite Hydrogel Substrate Fosters a Pro-Anabolic and Anti-Catabolic Microenvironment for Adherent Nucleus Pulposus Cells. (A) Schematic of the in vitro culture model where NPCs are seeded on different hydrogel formulations. (B) Histochemical staining for matrix deposition by NPCs cultured on different hydrogels, including Alcian Blue (top row) and Toluidine Blue (bottom row) staining. Scale bar: 200 μm. (C) Western blot analysis of anabolic (COL-2, ACAN) and catabolic (MMP-13, ADAMTS-4) protein markers in NPCs cultured on different hydrogel substrates. GAPDH served as a loading control. (D) Dot plot visualization of the relative protein expression levels from Fig. 3E The size and color intensity of each dot correspond to the expression level. (E) Western blot analysis of key proteins in the integrin-mediated signaling pathway (p-FAK, p-ERK, p-p38). (F) Dot plot visualization of the relative protein expression levels from Fig. 3E. (G) Western blot analysis of anabolic and catabolic markers in the presence of an integrin α2β1 blocking antibody (iα2β1). (H) Dot plot visualization of the relative protein expression levels from Fig. 3G. (I) Representative immunofluorescence images for Collagen II (green), MMP-13 (red), and Aggrecan (green) in NPCs cultured on different hydrogels. Cell nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. (J) Quantitative analysis of the relative fluorescence intensity for COL-2, MMP-13, and ACAN from the images in Fig. 3G. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

We next dissected the molecular drivers of this enhanced matrix deposition. Western blot analysis revealed that, compared to cells on the SAPH-R₁₆ substrate, NPCs on the SAPH-R₁₆GFOGER substrate exhibited a significant upregulation of the primary anabolic markers, Collagen Type II (COL-2) and Aggrecan (ACAN). This pro-anabolic effect was further amplified on the Mo₂C@SAPH-R₁₆GFOGER substrate. Concurrently, the expression of the critical catabolic enzymes, Matrix MMP-13 and ADAMTS-4, was most profoundly inhibited on the Mo₂C@SAPH-R₁₆GFOGER substrate (Fig. 3C). These trends were further illustrated in a graphical dot plot, where the size and color intensity of each spot corresponded to the relative protein expression, visually confirming that the composite hydrogel most effectively shifted the cellular metabolic balance towards a pro-regenerative, net anabolic state (Fig. 3D).

To further elucidate the mechanism by which the GFOGER motif promotes anabolism, we examined the integrin-mediated signaling pathway. As shown in Fig. 3E and F, Western blot analysis demonstrated that the phosphorylation levels of FAK, ERK, and p38 were significantly elevated in the SAPH-R₁₆GFOGER and Mo₂C@SAPH-R₁₆GFOGER groups compared to the non-functionalized SAPH-R₁₆ group. This indicates that the hydrogel activates the integrin/FAK/MAPK signaling axis to drive cellular functions.

To confirm the specificity of this mechanism, we introduced an integrin α2β1 blocking antibody (iα2β1) into the co-culture system. As expected, the addition of the blocking antibody significantly reversed the therapeutic effects of the composite hydrogel. The expression of anabolic markers (ACAN, COL-2) was markedly suppressed, while the catabolic markers (MMP-13, ADAMTS-4) were re-elevated, confirming that the regenerative effects are dependent on the specific interaction between the GFOGER motif and integrin α2β1 receptors (Fig. 3G, H).

These findings were further corroborated at the single-cell level by immunofluorescence staining. NPCs grown on the Mo₂C@SAPH-R₁₆GFOGER substrate exhibited the most intense fluorescent signals for both COL-2 and ACAN, coupled with a visibly diminished signal for MMP-13 (Fig. 3I). Quantitative analysis of the relative fluorescence intensity provided robust statistical support for these observations, revealing a significant upregulation of COL-2 and ACAN and a significant downregulation of MMP-13 in the composite hydrogel group compared to all other groups (Fig. 3J).Collectively, these multi-level data demonstrate that Mo₂C@SAPH-R₁₆GFOGER functions as a superior bioactive substrate, where pro-anabolic cues from the GFOGER motif and the low-oxidative-stress microenvironment created by Mo₂C synergistically instruct adherent NPCs to adopt a robustly pro-anabolic and anti-catabolic phenotype.

The Mo₂C@SAPH-R₁₆GFOGER hydrogel protects NPCs from oxidative stress and mitochondrial dysfunction

Having established the pro-regenerative properties of our hydrogel, we next investigated its capacity to protect nucleus pulposus cells (NPCs) from oxidative stress. To confirm that this protective effect was mediated by substances released from the scaffold, we utilized a Transwell co-culture system, physically separating the hydrogels from the NPCs (Fig. 4A). Tert-butyl hydroperoxide (TBHP) is a stable organic peroxide widely used to induce oxidative stress in vitro, as it initiates lipid peroxidation, thereby mimicking the IVDD microenvironment [42]. After inducing severe oxidative stress with TBHP, we observed a massive intracellular accumulation of multiple reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide anions (•O₂⁻), and hydroxyl radicals (•OH). Co-culture with the Mo₂C@SAPH-R₁₆GFOGER hydrogel demonstrated potent, broad-spectrum scavenging activity, significantly neutralizing all three ROS species to levels approaching the healthy control group (Fig. 4B, C). Flow cytometry analysis using a general ROS probe further confirmed that the composite hydrogel was the most effective treatment, markedly reducing the total intracellular ROS burden (Fig. 4D).

Fig. 4.

The Mo₂C@SAPH-R₁₆GFOGER Hydrogel Protects Nucleus Pulposus Cells from Oxidative Stress and Mitochondrial Dysfunction. (A) Schematic illustrating the Transwell co-culture system. (B) Representative fluorescence images for the detection of intracellular ROS, including H₂O₂, •O₂⁻, and •OH. Scale bar: 100 μm. (C) Corresponding quantitative analysis for the fluorescence intensity in Fig. 4B. (D) Flow cytometry analysis of total intracellular ROS levels in NPCs using the DCFH-DA probe. (E) Representative fluorescence images for the assessment of mitochondrial membrane potential (ΔΨm) via JC-1 staining. Scale bar: 100 μm. (F) Quantification of the red-to-green fluorescence ratio from Fig. 4E. (G) Volcano plot of lipidomic analysis showing differentially expressed lipid species between the TBHP and treatment groups. (H) Quantification of characteristic ferroptosis-related oxidized phosphatidylethanolamines (PE). (I) Representative TEM images of mitochondrial ultrastructure at low (top row) and high (bottom row) magnification. Scale bars: 1.5 μm (top), 500 nm (bottom). Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

Since mitochondria are a primary target of oxidative damage, we next investigated whether this robust ROS scavenging translated to the preservation of mitochondrial integrity. Oxidative stress triggers the collapse of the mitochondrial membrane potential (ΔΨm), a critical event initiating cell death. As expected, TBHP treatment induced severe mitochondrial depolarization in NPCs, evidenced by a shift from red (J-aggregates in healthy mitochondria) to predominantly green (J-monomers) fluorescence in the JC-1 assays (Fig. 4E, F). The collapse of the mitochondrial membrane potential is a critical point-of-no-return, leading to the release of pro-apoptotic factors and a halt in ATP synthesis [43].Co-culture with the Mo₂C@SAPH-R₁₆GFOGER hydrogel almost completely prevented this depolarization, maintaining a high red-to-green fluorescence ratio indicative of healthy, energized mitochondria. This potent preservation of ΔΨm was further corroborated using TMRE staining, which similarly showed that the fluorescence loss in TBHP-treated cells was significantly rescued by the Mo₂C@SAPH-R₁₆GFOGER hydrogel (Fig. S12).

To further characterize the specific impact on lipid peroxidation at the molecular level, we performed lipidomic profiling. The analysis revealed that the hydrogel treatment significantly reversed the accumulation of oxidized phosphatidylethanolamines (PE), which are key drivers of ferroptosis (Fig. 4G, H). This functional protection was mirrored at the structural level. TEM revealed that while TBHP caused severe mitochondrial damage, the observed ultrastructural changes—including mitochondrial shrinkage and the reduction or disappearance of internal cristae—are characteristic morphological hallmarks of ferroptosis. In stark contrast, the Mo₂C@SAPH-R₁₆GFOGER hydrogel preserved a healthy mitochondrial ultrastructure, with elongated morphology and well-defined, intact cristae, largely preserved a healthy ultrastructure similar to that of untreated controls (Fig. 4I). Collectively, these results demonstrate that the hydrogel exerts powerful cytoprotection by neutralizing oxidative stress, thereby preventing the downstream cascade of mitochondrial depolarization and structural damage. Having established that our hydrogel preserves the integrity of mitochondria—a central organelle in regulating iron metabolism and lipid peroxidation—we next sought to determine its direct capacity to inhibit the ferroptotic cell death pathway.

The Mo₂C@SAPH-R₁₆GFOGER hydrogel prevents ferroptosis in nucleus pulposus cells

To confirm that the antioxidant and mitochondrial-protective effects of the hydrogel translate to the inhibition of ferroptosis, we next directly assessed the key hallmarks of this cell death pathway. A central feature of ferroptosis is the accumulation of intracellular labile ferrous iron (Fe²⁺), which catalyzes lipid peroxidation. Using the Fe²⁺-specific fluorescent probe FerroOrange, we observed a massive increase in fluorescence intensity in NPCs treated with TBHP, indicating a severe iron overload. Co-culture with the Mo₂C@SAPH-R₁₆GFOGER hydrogel potently suppressed this iron accumulation, reducing fluorescence to near-control levels (Fig. 5A, B). This finding was corroborated by flow cytometry analysis, which likewise demonstrated a significant reduction in the FerroOrange-positive cell population in the composite hydrogel group (Fig. 5E).

Fig. 5.

The Mo₂C@SAPH-R₁₆GFOGER Hydrogel Inhibits Oxidative Stress-Induced Ferroptosis in Nucleus Pulposus Cells. (A) Representative fluorescence images of NPCs stained with the FerroOrange probe for the detection of intracellular labile iron (Fe²⁺). Scale bar: 100 μm. (B) Quantification of FerroOrange fluorescence intensity from Fig. 5A. (C) Representative images of NPCs stained with C11-BODIPY 581/591 for the detection of lipid peroxidation (LPO). Red channel (non-ox-C11) indicates unoxidized lipids, while the green channel (ox-C11) indicates peroxidized lipids. Scale bar: 100 μm. (D) Quantification of the ratio of green to red fluorescence intensity from Fig. 5C. (E) Flow cytometry analysis of intracellular Fe²⁺ using the FerroOrange probe. (F) Flow cytometry analysis of LPO using the C11-BODIPY probe. (G) Western blot analysis of key ferroptosis-regulating proteins: GPX4, FHC, SLC7A11, and ACSL4. GAPDH served as the loading control. (H) Dot plot visualization of the relative protein expression levels from Fig. 5G. The size and color intensity of each dot correspond to the expression level. (I) Immunofluorescence analysis showing representative images of GPX4 and SLC7A11 expression in NPCs. Scale bar: 100 μm. Data are presented as mean ± SD(n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

This iron overload is known to trigger rampant lipid peroxidation (LPO), the ultimate executioner of ferroptotic cell death. Using the ratiometric fluorescent probe C11-BODIPY, we observed a dramatic shift from red (unoxidized) to green (oxidized) fluorescence in TBHP-treated NPCs, signifying extensive LPO. The Mo₂C@SAPH-R₁₆GFOGER hydrogel almost completely abrogated this LPO, maintaining a high proportion of unoxidized lipids and confirming its ability to protect cell membranes from peroxidative damage (Fig. 5C, D). This was further validated by flow cytometry (Fig. 5F).

At the molecular level, this protection was linked to the restoration of the cell’s primary anti-ferroptotic machinery. Western blot analysis revealed that the hydrogel treatment rescued the expression of the master regulator GPX4 and its essential upstream transporter subunit SLC7A11, both of which were suppressed by TBHP. Furthermore, the hydrogel prevented the degradation of the iron-storage protein FHC and suppressed the upregulation of the pro-ferroptotic enzyme ACSL4 (Fig. 5G). A graphical dot plot visualization, where the size and intensity of each spot corresponded to the relative protein expression, further illustrated these protective effects (Fig. 5H). These findings were confirmed at the single-cell level by immunofluorescence, which showed a clear restoration of GPX4 and SLC7A11 protein levels in the Mo₂C@SAPH-R₁₆GFOGER group (Fig. 5I), with corresponding quantitative data provided in the supplementary materials (Fig. S13). To further confirm the specificity of this mechanism, we utilized the ferroptosis inducer RSL3. As shown in Figure S14, Mo₂C@SAPH-R₁₆GFOGER reversed RSL3-induced changes in GPX4, FHC, SLC7A11 and ACSL4 expression to a level comparable to the canonical inhibitor Ferrostatin-1, confirming that the hydrogel specifically targets the ferroptotic cascade. Additionally, we investigated the FSP1 pathway using the inhibitor iFSP1. The hydrogel maintained its ability to rescue ferroptosis-related proteins even in the presence of iFSP1 (Fig. S15), suggesting that its potent efficacy may be primarily mediated through the restoration of the canonical GSH-GPX4 axis rather than being solely dependent on the FSP1 pathway. Collectively, our assessments at both the cellular and molecular levels demonstrate that the Mo₂C@SAPH-R₁₆GFOGER hydrogel possesses potent antioxidant properties and effectively inhibits ferroptosis in nucleus pulposus cells.

Transcriptomic profiling confirms inhibition of ferroptosis and restoration of anabolic pathways

To elucidate the global transcriptomic changes underlying the protective effects of the Mo₂C@SAPH-R₁₆GFOGER hydrogel, we performed RNA-sequencing on nucleus pulposus cells (NPCs) treated with TBHP, both with and without co-culture with the therapeutic hydrogel. The volcano plot of differentially expressed genes (DEGs) revealed that the hydrogel treatment induced profound transcriptomic shifts, rescuing the pathological gene expression profile induced by TBHP. Notably, key genes associated with extracellular matrix (ECM) synthesis, including Aggrecan (ACAN) and Collagen Type II Alpha 1 Chain (COL2A1), were significantly upregulated by the hydrogel treatment. Conversely, genes promoting ECM degradation (MMP13), ferroptosis (ACSL4, GLS2), and apoptosis (BCL2L11) were all significantly downregulated (Fig. 6A). Hierarchical clustering of all DEGs demonstrated a clear and distinct separation between the TBHP-treated group and the hydrogel-protected group, with two major clusters of genes being reciprocally regulated, confirming a distinct and widespread therapeutic effect at the transcriptomic level (Fig. 6B). This unbiased, genome-wide view strongly suggests that the hydrogel not only counteracts cell death but also actively promotes a pro-regenerative cellular state.

Fig. 6.

Transcriptomic Analysis of the Regulatory Effects of the Mo₂C@SAPH-R₁₆GFOGER Hydrogel. (A) Volcano plot of differentially expressed genes (DEGs) in NPCs. (B) Heatmap illustrating the hierarchical clustering of DEGs. (C) Gene Set Enrichment Analysis (GSEA) plot for the Glutathione Metabolism pathway in the TBHP vs. Control condition. (D) GSEA plot for the Glutathione Metabolism pathway in the Mo₂C@SAPH-R₁₆GFOGER vs. TBHP condition. (E) GSEA plot for the Ferroptosis pathway in the Mo₂C@SAPH-R₁₆GFOGER vs. TBHP condition. (F) GSEA plot for the Apoptosis pathway in the Mo₂C@SAPH-R₁₆GFOGER vs. TBHP condition. (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. (H) Gene Ontology (GO) enrichment analysis of DEGs, categorized by Biological Process, Cellular Component, and Molecular Function

To identify the key biological processes modulated by the hydrogel, we performed Gene Set Enrichment Analysis (GSEA). As a baseline, TBHP treatment alone was found to induce a significant cellular stress response, evidenced by the positive enrichment of the “Glutathione metabolism” pathway as cells attempted to mount an antioxidant defense (Fig. 6C). In contrast, co-culture with the Mo₂C@SAPH-R₁₆GFOGER hydrogel led to a significant negative enrichment of this pathway relative to the stressed state, indicating that the hydrogel’s potent ROS-scavenging activity alleviated the oxidative burden and restored cellular redox homeostasis (Fig. 6D). More importantly, GSEA confirmed that the treatment led to a strong and significant negative enrichment of gene sets associated with cell death, most notably “Ferroptosis” and “Apoptosis” (Fig. 6E, F). Broader pathway analyses using Kyoto Encyclopedia of Genes and Genomes (KEGG) corroborated these findings, highlighting “Ferroptosis,” “Apoptosis,” the “p53 signaling pathway,” and “ECM-receptor interaction” among the most significantly modulated pathways by the treatment (Fig. 6G). Notably, the treatment significantly modulated terms related to glutathione biosynthetic and metabolic processes, the apoptotic process, mitochondrion organization, and cell-matrix adhesion, directly aligning with the hydrogel’s observed antioxidant, anti-apoptotic, and pro-regenerative functions (Fig. 6H).

To extract deeper mechanistic insights from the transcriptomic data, we performed further upstream regulator analysis and Weighted Gene Co-expression Network Analysis (WGCNA). Gene Set Enrichment Analysis (GSEA) identified the p53 signaling pathway as a master regulator significantly downregulated by the hydrogel treatment (Fig. S16), which aligns with its role in orchestrating both cell death and stress responses. Furthermore, WGCNA identified a distinct gene module (the Blue module) that showed a strong positive correlation with the TBHP-induced degenerative state while being negatively correlated with the treatment group (Fig. S17). KEGG analysis of this specific module revealed a significant co-enrichment of “Ferroptosis”, “ECM-receptor interaction”, and “Focal adhesion” pathways (Fig. S18). This finding suggests that under oxidative stress, the activation of cell death programs is tightly coupled with pathological ECM dysregulation and the disruption of cell-matrix adhesion signaling. The Mo₂C@SAPH-R₁₆GFOGER hydrogel exerts its therapeutic effect by suppressing this entire pathological gene module, thereby simultaneously blocking ferroptosis and restoring ECM homeostasis.

The hydrogel co-inhibits ferroptosis and apoptosis by suppressing oxidative stress and the p53 signaling pathway

Ferroptosis is a novel form of cell death caused by iron-dependent lipid peroxidation, where severe iron overload and peroxidation ultimately trigger cell death. To elucidate the intrinsic mechanism by which the hydrogel inhibits cell death, we first focused on the ferroptosis pathway. As ferroptosis is driven by oxidative stress, we assessed key upstream indicators. MitoSOX staining revealed that TBHP treatment induced a dramatic accumulation of mitochondrial superoxide, whereas the Mo₂C@SAPH-R₁₆GFOGER hydrogel was able to effectively scavenge these ROS, indicating that it can significantly ameliorate mitochondrial oxidative stress (Fig. 7A, B). Concurrently, the primary intracellular antioxidant, GSH, was severely depleted following TBHP treatment, but the hydrogel treatment successfully restored GSH levels, thereby maintaining cellular redox homeostasis (Fig. 7C). These results confirm that the hydrogel can effectively block the upstream oxidative damage required for the initiation of ferroptosis.

Fig. 7.

The Mo₂C@SAPH-R₁₆GFOGER Hydrogel Inhibits Apoptosis and Ferroptosis by Targeting the Mitochondrial ROS/p53 Axis. (A) Representative fluorescence images of the MitoSOX probe for the detection of mitochondrial superoxide (red). Scale bar: 100 μm. (B) Quantification of MitoSOX fluorescence intensity from Fig. 7A. (C) Quantification of intracellular glutathione (GSH) concentration. (D) Representative fluorescence images of the TUNEL assay for the detection of DNA fragmentation (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. (E) Quantification of TUNEL fluorescence intensity from Fig. 7D. (F) Flow cytometry analysis of apoptosis in NPCs using an Annexin V-FITC/PI staining kit. (G) Western blot analysis of key proteins in the p53 signaling pathway (p-P53, P53) and downstream effectors of apoptosis (BCL2L11, CASP7) and ferroptosis (GLS2). (H) Dot plot visualization of the relative protein expression levels from Fig. 7G. The color intensity of each dot corresponds to the expression level. (I) Schematic diagram summarizing the comprehensive cytoprotective mechanism of the Mo₂C@SAPH-R₁₆GFOGER hydrogel. Data are presented as mean ± SD(n = 3). ***p < 0.001, ****p < 0.0001; ns, not significant

Intriguingly, our transcriptomic analysis also revealed that the hydrogel significantly suppressed the apoptosis pathway (Fig. 6F). To validate this finding, we performed apoptosis assays. TUNEL staining visually demonstrated that DNA fragmentation, a late marker of apoptosis, was significantly reduced in the hydrogel-treated group (Fig. 7D), and quantitative analysis confirmed a significant decrease in the proportion of apoptotic cells (Fig. 7E). Correspondingly, flow cytometry analysis yielded consistent results, proving that the hydrogel effectively protects cells from apoptosis (Fig. 7F).

To identify a common molecular node linking the ferroptosis and apoptosis pathways, we investigated the p53 signaling pathway, which was notably enriched in our transcriptomic analysis (Fig. 6G). As a central stress sensor, p53 can regulate both modes of cell death. Western blot results confirmed that by mitigating upstream oxidative stress, the hydrogel effectively inhibited the phosphorylation and activation of p53. The downregulation of p53 activity, in turn, suppressed the expression of its pro-ferroptotic target, GLS2, while simultaneously inhibiting its pro-apoptotic targets, BCL2L11 (BIM) and the executioner Caspase-7 (Fig. 7G, H). As summarized in the mechanistic diagram (Fig. 7I), these results demonstrate that the hydrogel exerts a comprehensive cytoprotective effect by targeting the p53 signaling hub to simultaneously inhibit both apoptotic and ferroptotic cell death pathways.

Injectable Mo₂C@SAPH-R₁₆GFOGER hydrogel alleviates IVDD progression and promotes tissue regeneration in vivo

To evaluate the therapeutic potential of the Mo₂C@SAPH-R₁₆GFOGER hydrogel in a clinically relevant setting, we established a needle puncture-induced intervertebral disc degeneration (IVDD) model in rats. A single intra-discal injection of the hydrogel was administered, and therapeutic efficacy was monitored over eight weeks (Fig. 8A, B). Histological analysis of major organs (heart, liver, spleen, lungs, and kidney) at 4 and 8 weeks revealed no signs of inflammation or pathological damage, confirming the high in vivo biocompatibility of the materials (Fig. S19). Mechanistically, Mo₂C nanosheets degrade into biocompatible molybdate ions (MoO₄²⁻), which are water-soluble and efficiently excreted via the renal system [44, 45]. This metabolic pathway prevents long-term accumulation, consistent with the absence of chronic toxicity observed in our 8-week evaluation. With safety established, we assessed therapeutic outcomes. X-ray analysis showed that while the IVDD group suffered a severe, progressive loss of disc height, the Mo₂C@SAPH-R₁₆GFOGER hydrogel was the most effective treatment, significantly attenuating the decrease in the disc height index (DHI) (Fig. 8C, E). These structural findings were supported by T2-weighted MRI. The composite hydrogel markedly preserved the high signal intensity of the nucleus pulposus, preventing the “black disc” appearance characteristic of severe degeneration and resulting in the lowest (healthiest) Pfirrmann grades (Fig. 8D, F). This preservation of a low Pfirrmann grade in the treated group is a indicator of successful therapeutic intervention, as it reflects the maintenance of both tissue hydration and structural integrity [46].

Fig. 8.

The Mo₂C@SAPH-R₁₆GFOGER Hydrogel Attenuates IVDD Progression and Inhibits Ferroptosis In Vivo. (A) Photograph of the intra-discal injection procedure. (B) Schematic of the in vivo experimental design. (C) Representative X-ray images of rat tail intervertebral discs after 4 weeks and 8 weeks. (D) Representative T2-weighted MRI scans of the discs after 4 weeks and 8 weeks. (E) Quantitative analysis of the disc height index (DHI). (F) Quantitative analysis of the Pfirrmann grade. (G) Immunohistochemical staining for ACAN, COL-2, and ACSL4 in disc tissues at 8 weeks. Scale bar: 200 μm. (H)Immunofluorescence staining for GPX4 in disc tissues at 8 weeks. Scale bar: 200 μm. (I) Quantitative analysis of glycosaminoglycan (GAG) content using DMMB assay. (J) Photograph of the biomechanical compression testing setup. (K) Quantitative analysis of the compressive modulus of the motion segments. Data are presented as mean ± SD(n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant

At the molecular level, immunohistochemical and immunofluorescence analyses at 8 weeks confirmed these macroscopic findings. The composite hydrogel powerfully promoted ECM regeneration, evidenced by the robust restoration of ACAN and COL-2 expression. Critically, we validated the inhibition of ferroptosis in vivo, as the hydrogel led to a significant suppression of the pro-ferroptotic marker ACSL4 and a corresponding restoration of the key protective enzyme GPX4 (Fig. 8G, H). Biochemical and biomechanical assessments provided further evidence of functional regeneration. The glycosaminoglycan (GAG) content, a critical component of the NP matrix, was quantitatively measured using a DMMB assay. The composite hydrogel group exhibited a significantly higher GAG level than the IVDD group (Fig. 8I). Moreover, functional restoration was evaluated through biomechanical testing of the spinal motion segments (Fig. 8J). The compressive modulus in the Mo₂C@SAPH-R₁₆GFOGER group was significantly higher than that of the IVDD group, indicating the robust recovery of the disc’s mechanical support capability (Fig. 8K).

Histological analysis further confirmed a profound restoration of tissue architecture in the Mo₂C@SAPH-R₁₆GFOGER group. Macroscopic examination of the dissected discs revealed a larger, more gelatinous nucleus pulposus (NP) compared to the fibrotic and collapsed appearance of the IVDD group (Fig. 9A). Hematoxylin and Eosin (H&E) staining confirmed that the composite hydrogel preserved a well-organized tissue structure, maintaining a healthy, cellular NP and a well-defined lamellar structure in the anulus fibrosus (AF) (Fig. 9B). The preservation of the NP’s critical biochemical composition was confirmed by multiple proteoglycan-detecting stains. The Mo₂C@SAPH-R₁₆GFOGER group demonstrated remarkable preservation of the proteoglycan-rich matrix, with intense and uniform red (Safranin O) and purple (Toluidine Blue) staining throughout the NP (Fig. 9C, D). This finding was further corroborated by intense Alcian Blue staining (Fig. S20). A comprehensive histological scoring system quantitatively confirmed that the composite hydrogel group had the lowest (best) degeneration scores across all parameters (Fig. 9E). Taken together, the data from our rat model of intervertebral disc injury demonstrate that the Mo₂C@SAPH-R₁₆GFOGER hydrogel exerts a potent therapeutic effect in vivo and holds considerable potential for clinical translation.

Fig. 9.

The Mo₂C@SAPH-R₁₆GFOGER Hydrogel Promotes Histological Regeneration of the Intervertebral Disc. (A) Macroscopic images of dissected discs after 4 weeks and 8 weeks. Scale bar: 1 mm. (B) Hematoxylin and Eosin (H&E) staining. Scale bar: 1 mm. (C) Safranin O-Fast Green staining. Scale bar: 1 mm. (D) Toluidine Blue staining. Scale bar: 1 mm. (E) Heatmap of the comprehensive histological scores after 4 weeks and 8 weeks

Finally, it is important to acknowledge the limitations of the current study. First, we did not employ ferroptosis-specific genetic models (e.g., GPX4 knockout mice) to establish strict genetic causality, although our pharmacological data strongly implicate ferroptosis as a key driver. Second, while the rat tail puncture model is a well-established system for IVDD, it differs from human lumbar IVDD in terms of mechanical loading, nutritional environment, and anatomical scale. Therefore, further preclinical studies in large animals and investigations utilizing genetic tools are warranted to validate and optimize the proposed therapeutic strategy before clinical application.

Conclusion

In this study, we developed a multifunctional, injectable composite nanogel, Mo₂C@SAPH-R₁₆GFOGER, for the treatment of intervertebral disc degeneration. We established that the progressive degeneration of human intervertebral discs is closely associated with the molecular hallmarks of ferroptosis, ferroptosis is a key regulatory node rather than a passive marker. Our composite nanogel leverages the synergistic effects of a biomimetic peptide scaffold (SAPH-R₁₆GFOGER) that promotes ECM synthesis and an antioxidant nanomaterial (Mo₂C) that scavenges ROS. We demonstrated that the nanogel effectively protects nucleus pulposus cells from oxidative stress by preserving mitochondrial integrity and comprehensively inhibiting both ferroptosis and apoptosis. Current ferroptosis-targeting strategies typically rely on the local delivery of small molecule inhibitors (e.g., Ferrostatin-1) or iron chelators. These approaches often suffer from rapid clearance and provide no physical support to the degenerated disc. Our supramolecular hydrogel serves a dual function: it acts as a sustained depot for the anti-ferroptotic agent (Mo₂C) to ensure prolonged retention (> 3 weeks) and provides immediate biomechanical support to the disc space via hydrostatic pressure. In addition, our system integrates the catalytic activity of Mo₂C with a specific bioactive ligand (GFOGER). Standard antioxidant scaffolds often lack the specific biological cues required to restart extracellular matrix (ECM) anabolism once oxidative stress is reduced. However, the GFOGER motif is essential for engaging Integrin α2β1 to promote collagen synthesis, a functional recovery that generic antioxidant scaffolds do not actively drive. In a preclinical rat model of IVDD, a single injection of the nanogel successfully attenuated disc degeneration, preserved tissue structure and hydration, and promoted matrix regeneration by inhibiting ferroptosis in vivo. These findings highlight that synergistically targeting multiple cell death pathways and promoting anabolism with an advanced, multifunctional biomaterial is a promising strategy for the clinical treatment of IVDD.

Experimental section (Materials and Methods)

Materials

Unless otherwise specified, all chemical reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F12), and penicillin-streptomycin were purchased from Gibco (Grand Island, NY, USA). The Cell Counting Kit-8 (CCK-8), TUNEL Apoptosis Assay Kit, DCFH-DA, MitoSOX™ Red, and BODIPY™ 581/591 C11 were obtained from Beyotime (Shanghai, China). The Annexin V-FITC/PI kit was from Vazyme Biotech (Nanjing, China). Crystal Violet and JC-1 dye were from Solarbio (Beijing, China). The FerroOrange probe was from Dojindo (Kumamoto, Japan). Unless stated otherwise, primary and secondary antibodies were purchased from ProteinTech (Wuhan, China).Molybdenum carbide (Mo₂C) nanosheets were prepared as described previously [45] [Ultra-high antioxidant, anti-apoptosis and pro-angiogenesis effect of Mo₂C nanosheets for treatment of diabetic wounds. Nano Today 55 (2024) 102202.].

Synthesis and characterization of the injectable hydrogel

The functionalized R₁₆GFOGER peptide was synthesized using standard solid-phase peptide synthesis (SPPS). The final product was purified by reverse-phase HPLC and its molecular weight was confirmed by MS. The Mo₂C@SAPH-R₁₆GFOGER hydrogel was prepared by dissolving the R₁₆GFOGER peptide powder in sterile deionized water to a final concentration of 2% (w/v). Mo₂C nanosheets were then dispersed into the peptide solution to a final concentration of 30 µg/mL and sonicated briefly to ensure uniform distribution. The precursor solution was stored at 4 °C before use and allowed to self-assemble into a hydrogel by incubation at 37 °C for 10 min.

The nanostructure of the self-assembled hydrogels was observed Transmission TEM after negative staining with phosphotungstic acid. The microarchitecture of lyophilized hydrogel scaffolds was imaged using SEM after sputter-coating with gold. The rheological properties were measured using a rheometer with parallel plates. Strain sweep tests were performed to determine the linear viscoelastic region and compare the storage modulus (G’) and loss modulus (G’’). Cyclical strain tests (alternating between 1% and 20% strain) were performed to evaluate the shear-thinning and self-healing properties.

Collection of human intervertebral disc samples

Human NP tissue samples were procured from patients at the Second Affiliated Hospital of Wenzhou Medical University with the approval of the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China; approval number: 2024-K-207-01). All patients provided written, informed consent prior to the procedure. Degenerative disc samples of Pfirrmann grades II, III, IV, and V were collected based on T2-weighted MRI scans (n = 5 per group; mean age, 54 years; age range: 45–65 years). For comparative analysis, Grade II samples were used as the less-degenerated baseline control.

Cell culture and treatments

NPCs were isolated from the NP tissues of Sprague–Dawley rats. Tissues were minced and digested with 2 mg mL⁻¹ collagenase II for 5 h at 37 °C. Isolated NPCs were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37 °C with 5% CO₂. The culture medium was refreshed every 2 days. To induce oxidative stress, the culture medium was replaced with fresh medium containing 100 µM tert-Butyl hydroperoxide (TBHP) simultaneously with the indicated treatments (PBS, free Mo₂C). The cells were then co-incubated for 8 h. For experiments utilizing the Transwell system, the hydrogels were placed in the upper insert while the cells in the lower well were treated with TBHP-containing medium for the same duration.

In vitro assays

Cytocompatibility, Viability, and proliferation assays

NPCs were plated in 96-well plates at a density of 8 × 10³ cells per well. After treatment, cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions (Beyotime, China). For cytocompatibility, Live/Dead staining was performed using calcein-AM and propidium iodide (PI) after 24 h of co-culture (Beyotime, Shanghai China). For proliferation/biomass assessment, cells cultured on hydrogels for 72 h were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet solution (Solarbio, Beijing China). After washing, the retained dye was dissolved with 10% acetic acid and the absorbance was measured at 590 nm.

Histochemical staining

After 7 days of co-culture with the hydrogels, cells were fixed with 4% paraformaldehyde for 30 min and stained with either Alcian blue solution for 1 h or 0.1% Toluidine Blue solution for 30 min to detect glycosaminoglycan (GAG) deposition (Beyotime, Shanghai China). Images were captured using an Olympus microscope (Olympus Inc., Japan) and staining intensity was quantified using ImageJ software.

Intracellular and mitochondrial ROS detection

NPCs were seeded in 96-well plates. After treatment, cells were incubated with 10 µM DCFH-DA for 30 min at 37 °C to detect total intracellular ROS (Beyotime, Shanghai China). To detect mitochondrial superoxide, cells were incubated with 5 µM MitoSOX™ Red reagent (Beyotime, Shanghai China) for 30 min at 37 °C. Fluorescence images were captured using an Olympus microscope.

Lipid peroxidation and iron assay

Lipid peroxidation was assessed using the fluorescent probe BODIPY™ 581/591 C11 (2 µM, 1 h incubation, Beyotime, Shanghai China). The ratio of green (oxidized) to red (reduced) fluorescence was quantified to determine the level of lipid peroxidation. Cellular Fe²⁺ levels were detected using the FerroOrange probe (1 µM, 30 min incubation, Dojindo, Kumamoto, Japan), which fluoresces upon binding to intracellular labile iron.

Mitochondrial assays

Mitochondrial membrane potential was assessed using JC-1 dye (5 µM, 20 min incubation, Solarbio, Beijing China) and Tetramethylrhodamine, Ethyl Ester (TMRE) (100 nM, 30 min incubation; Invitrogen, USA). For JC-1, a high red/green fluorescence ratio indicates healthy, polarized mitochondria. Mitochondrial structure was observed using a HITACHI HT7700 transmission electron microscope after standard sample preparation including fixation in glutaraldehyde, post-fixation in osmium tetroxide, serial dehydration in ethanol, and embedding in resin. To visualize cellular internalization, Bio-Transmission Electron Microscopy (Bio-TEM) was performed. NPCs were incubated with Mo₂C@SAPH-R₁₆GFOGER for 24 h, fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, serially dehydrated, and embedded in resin. Ultrathin Sect.  (70 nm) were stained and imaged using a Hitachi HT7700 TEM.

Apoptosis assays

Apoptosis was quantified using an Annexin V-FITC/PI Apoptosis Detection Kit and analyzed via CytoFLEX flow cytometry (Vazyme Biotech, China). Annexin V-positive/PI-negative cells were identified as early apoptotic, while Annexin V-positive/PI-positive cells were identified as late apoptotic. DNA fragmentation was detected using a TUNEL assay kit, with DAPI used for nuclear counterstaining (Solarbio, Beijing China).

Western blotting

Following treatment, total protein was extracted from NPCs using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (20 µg) per sample were separated by 8–12% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, California, USA). Membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4 °C with primary antibodies against: ACAN, ADAMTS-4, COL-II, MMP-13, GPX4, FHC, p-p53, p53, GLS2, BCL2L11, and CASP7 (1:1000; ProteinTech, Wuhan, China). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000; ProteinTech, Wuhan, China) for 1.5 h at room temperature. Protein bands were visualized using an ECL luminescence reagent and the band intensity was quantified using ImageJ software.

Immunofluorescence staining

NPCs were seeded on glass coverslips in 12-well plates. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h. Cells were then incubated overnight at 4 °C with primary antibodies against MMP-13, COL-II, ACAN, GPX4 and SLC7A11 (1:100; ProteinTech, Wuhan, China). Subsequently, cells were incubated with Alexa Fluor-conjugated secondary antibodies (1:500, ProteinTech, Wuhan, China) for 2 h at room temperature. Nuclei were counterstained with DAPI (Servicebio, China), and images were captured via fluorescence microscopy. Fluorescence intensity was quantified using ImageJ software.

RNA-Sequencing and bioinformatic analysis

Total RNA was extracted from cultured rat nucleus pulposus cells (NPCs) after the indicated treatments using a RNeasy Mini Kit (Qiagen, Germany). RNA quality and quantity were assessed to ensure high quality (OD260/280 = 1.8–2.2, RIN ≥ 6.5). Strand-specific libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina NovaSeq 6000 platform. Raw data underwent quality assessment via FastQC, and subsequent bioinformatic analyses, including differential gene expression and gene set enrichment analysis (GSEA), were performed. Weighted Gene Co-expression Network Analysis (WGCNA) was conducted using the “WGCNA” R package to identify key gene modules correlated with experimental traits, followed by KEGG enrichment analysis. For lipidomics profiling, lipids were extracted from NPCs using the MTBE method and analyzed via Liquid Chromatography-Mass Spectrometry (LC-MS). The raw data were processed for peak alignment and identification. Oxidized phosphatidylethanolamine (PE) species, which are characteristic of ferroptosis, were specifically targeted for differential expression analysis.

Animal model and in vivo experiments

All animal procedures were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University (No. WIUCAS24051301). Sprague-Dawley rats (male, 250–300 g) were used. After anesthesia with an intraperitoneal injection of pentobarbital, IVDD was induced by puncturing the Co7/Co8 caudal intervertebral disc with a 22G needle. A single 5 µL injection was administered into the center of the disc using a microinjector. The rats were divided into five groups (n = 5 per group): (1) Sham Control (no injection), (2) IVDD + PBS, (3) IVDD + free Mo₂C, (4) IVDD + SAPH-R₁₆GFOGER, and (5) IVDD + Mo₂C@SAPH-R₁₆GFOGER.

In vivo evaluation: Imaging, Histology and function

For in vivo retention studies, Mo₂C nanoparticles were labeled with fluorescein isothiocyanate (FITC) before incorporation into the hydrogel. After injection, rats were imaged at 0, 1, 2, and 3 weeks using an in vivo imaging system (IVIS). At 4 weeks and 8 weeks post-operation, rats were subjected to X-ray (Kubtec, KUB Technologies Inc.) and 3.0T MRI (Philips Intera Achieva 3.0 MR) scans to evaluate disc height and hydration. The Disc Height Index (DHI) and Pfirrmann grading system were used for quantitative analysis. Following imaging, rats were euthanized. For functional assessment, fresh spinal motion segments were harvested for biomechanical testing. Samples were potted in PMMA and subjected to unconfined compression (0.5 mm/min) using an Instron machine to calculate the compressive modulus. Additionally, the GAG content of the NP was quantified using a DMMB assay. Specimens were fixed in 4% formaldehyde for 48 h, decalcified in 10% EDTA for one month, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with Hematoxylin and Eosin (H&E), Safranin-O/Fast Green (SO&FG), Alcian Blue, and Toluidine Blue. Immunohistochemistry and immunofluorescence were performed to analyze protein expression in vivo.

For comprehensive safety evaluation, a 4-week longitudinal study was conducted. Serum biochemical markers (ALT, AST, BUN, CRE) and inflammatory cytokines were monitored weekly. At the endpoint, major organs (heart, liver, spleen, lung, kidney, brain) were harvested for ex vivo fluorescence imaging to evaluate biodistribution. Additionally, major organs were collected at 4 and 8 weeks and processed for H&E staining to detect any potential systemic pathological damage.

Statistical analysis

All quantitative data are expressed as mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using Prism 9 software (GraphPad Software, USA). Comparisons between multiple groups were made using a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. *, p-value < 0.05, were considered statistically significant, with increased confidence levels shown as **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Some of the diagrams were created with BioRender.

Author contributions

G.L., Z.G., and Z.C. contributed equally to this work. G.L., Z.G., Z.C., and X.Z. conceived and designed the study. G.L., Z.G., Q.Z., and X.L. performed the experiments and data collection. Z.C., G.L., and Z.G. analyzed the data. G.L., Z.C., H.L., S.L., and X.Z. contributed to the methodology. Y.C., S.Y., and S.L. developed the software. S.S. and X.Z.W. participated in data validation and visualization. G.L. and Z.G. wrote the original draft. X.Z., Z.C., and G.L. reviewed and edited the manuscript. A.W. supervised the project. X.Y.W., X.Z., X.G., and Y.Z. acquired funding. A.W. and X.Y.W. provided resources. All authors discussed the results and approved the final manuscript.

Funding

This work is supported by National Natural Science Foundation of China (82372461, 82172494, 22472042), National Key Research and Development Program of China (2025YFC2428200), Zhejiang Provincial Science and Technology Project for Public Welfare (No. LQ24H090010), Wenzhou Science and Technology Bureau Foundation (ZY2023015), High-level Innovation Team of Wenzhou’s “Ouyue Talent Plan” (No. 2024R3003) and Clinical Medicine Plus X - Scholars Project of the Second Affiliated Hospital of Wenzhou Medical University.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Human NP tissue samples were procured from patients at the Second Affiliated Hospital of Wenzhou Medical University with the approval of the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China; approval number: 2024-K-207-01). The procedures and treatment were approved by the Experimental Animal Ethics Committee of Wenzhou Institute, University of Chinese Academy of Sciences (Issue No. WIUCAS24051301).

Competing interests

The authors declare no competing interests.