Cuproptosis and Orthopedic Diseases: Molecular Mechanisms of Copper Metabolic Imbalance in the Skeletal System and Clinical Translation Prospects

Abstract

Copper, an essential trace element, serves as a critical cofactor for enzymes governing energy metabolism, antioxidant defense, and connective tissue synthesis. The narrow margin between physiological requirement and toxicity necessitates precise homeostatic control, as both deficiency and excess compromise bone cell viability. Dysregulated copper metabolism has emerged as a pathogenic driver in orthopedic disorders, prompting investigation of cuproptosis—a distinctive form of mitochondrial cell death triggered by copper overload. The development of numerous diseases may occur when copper levels are abnormal in the body. A newly proposed form of cell death, known as cuproptosis, is distinct from necroptosis, apoptosis, autophagy, ferroptosis, and pyroptosis due to its cause: an excessive accumulation of intracellular copper. In the TCA cycle, copper binds to lipoylated proteins, which induces their aggregation, proteotoxic stress, the loss of Fe-S cluster proteins, and ultimately results in cell death. This process highlights the intricate relationship between metal homeostasis and cellular metabolism illustrating excess copper can induce cellular toxicity through biochemical mechanisms. Ever since cuproptosis was introduced, increasing research has highlighted its crucial involvement in the occurrence, development and management of many orthopedic disorders. The skeletal system presents unique features that make it particularly relevant for cuproptosis research: (i) bone tissue undergoes continuous remodeling with high metabolic demands, making it highly sensitive to mitochondrial dysfunction; (ii) osteoblasts, osteoclasts, and chondrocytes rely heavily on TCA cycle activity for energy production and differentiation; (iii) copper-containing biomaterials have been widely used in orthopedics for their dual osteogenic and antimicrobial properties, yet their mechanisms remained unclear until the discovery of cuproptosis; and (iv) the hypoxic microenvironment in bone marrow and inflammatory joints creates conditions favorable for copper accumulation and lipoylated protein aggregation. These distinctive characteristics position orthopedic diseases as an ideal model system for investigating cuproptosis pathophysiology and its therapeutic applications. In the review, we summarize copper homeostasis, copper ions’ role in the major cells of bone metabolism, and the interconnections of cuproptosis in osteosarcoma, osteoarthritis, rheumatoid arthritis, osteoporosis, osteomyelitis, spinal disorders, implant infections, and wound repair. We hope to provide novel diagnostic and therapeutic ideas for these diseases by modulating copper homeostasis and targeting cuproptosis.

Introduction

Copper is a metal element with dichotomous effects on cellular functions.¹ This duality is particularly evident in skeletal biology: while copper excess induces cuproptosis and compromises bone cell viability, copper deficiency equally jeopardizes skeletal integrity through distinct mechanisms. Copper serves as an essential cofactor for lysyl oxidase (LOX), the enzyme responsible for collagen and elastin cross-linking—critical processes for bone matrix strength and resilience. Classic studies demonstrate that copper-deficient diets cause LOX dysfunction, resulting in defective collagen cross-linking, skeletal abnormalities, and connective tissue fragility in animal models.² Clinically, Menkes disease—a congenital copper deficiency disorder—manifests with severe osteoporosis, skeletal dysplasia, and pathological fractures due to impaired LOX activity and collagen maturation.³ These findings establish that copper operates within a narrow therapeutic window in bone: deficiency compromises structural matrix integrity via LOX impairment, whereas excess triggers mitochondrial dysfunction via cuproptosis. Cells require optimal copper concentrations for survival: copper deficiency disrupts cellular respiration and metabolism, whereas excessive copper impairs cell viability and can lead to cell death.⁴ As a structural component and cofactor of numerous enzymes, copper regulates various cellular processes including biosynthesis, respiration, and oxidative defense, and its homeostatic imbalance significantly compromises cellular function.^5,6 Due to the cytotoxicity of copper, cells maintain low intracellular copper levels through tightly regulated uptake, efflux, and distribution mechanisms.⁷ Dysregulation of copper homeostasis is implicated in multiple pathological conditions, including cardiovascular diseases, Menkes disease, neurodegenerative disorders, Wilson’s disease, and cancer.⁸ Cuproptosis is a form of regulated cell death triggered by the direct binding of copper ions to lipoylated proteins (such as DLAT) in the tricarboxylic acid (TCA) cycle. This binding induces aggregation of lipoylated proteins and loss of iron-sulfur cluster proteins, ultimately leading to cell death through proteotoxic stress.⁹ Studies have demonstrated that copper homeostasis disruption impairs the development of bone, cartilage, and synovium by affecting osteoblasts, osteoclasts, chondrocytes, and synoviocytes.¹⁰ Both copper deficiency and excess can lead to abnormal bone metabolism. Dietary copper deficiency, reduced serum copper levels, and decreased copper in local tissues result in decreased bone mineral density (BMD) and altered bone structure, whereas copper excess exerts toxic effects on bone tissue or reduces bone strength by influencing lipid peroxidation, thereby increasing fracture incidence.¹¹ Copper participates in antioxidant defense, mitochondrial respiration, and ferroxidase activity through the activation of various transcription factors, processes that are essential for iron absorption, bone formation, osteoclast differentiation, and metabolic homeostasis maintenance. Normal copper homeostasis constitutes the foundation for maintaining musculoskeletal health and managing bone and joint-related diseases.¹² Copper stimulates osteoblast activity and proliferation by upregulating the expression of critical bone-related genes, including OCN (osteocalcin), OPN (osteopontin), ALP (alkaline phosphatase), Col1 (type I collagen), and VEGF (vascular endothelial growth factor).¹³ The incorporation of copper into biomaterials significantly promotes osteoblast proliferation, thereby substantially enhancing bone formation and bone defect repair.^14,15 Copper-loaded MgO nanoparticles inhibit osteoclast formation in a dose-dependent manner.¹⁶ Copper at 50μM reduces the proliferation rate of mesenchymal stem cells (MSCs) but enhances their differentiation capacity toward the osteogenic lineage.¹⁷ MSCs exhibit greater tolerance to high copper concentrations, with adverse effects observed only when Cu²⁺ concentrations exceed 250μM. Copper stimulates endothelial cell proliferation and migration by enhancing VEGF expression, thereby promoting neovascularization.¹⁸ The integration of copper into biomaterials reduces pro-inflammatory cytokine gene expression by suppressing M1 macrophage formation while increasing anti-inflammatory factor expression through the promotion of M2 macrophage differentiation and proliferation.^19,20 Cuproptosis-related genes (CRGs) are differentially expressed in osteoarthritis, rheumatoid arthritis, osteosarcoma, osteoporosis, ankylosing spondylitis, and lumbar disc herniation, whereas copper-loaded biomaterials can treat orthopedic infectious diseases by inducing cuproptosis. These findings provide promise and confidence for elucidating the relationship between cuproptosis and orthopedic diseases.

Copper Metabolism and Homeostasis

Copper is primarily absorbed from the diet through the duodenum and jejunum, which constitute critical components of the digestive system.²¹ At the apical membrane of enterocytes, STEAP (six-transmembrane epithelial antigen of the prostate) proteins reduce divalent copper (Cu²⁺) to monovalent copper (Cu⁺), after which the plasma membrane copper transporter CTR1 (SLC31A1) mediates the transport of Cu⁺ from the intestinal lumen into enterocytes.^22,23 Following gastrointestinal absorption, copper (Cu) is released into the bloodstream primarily through ATP7A, with ATP7B also contributing partially to this process. In the blood, copper binds to soluble chaperone proteins, including histidine, albumin, transcuprein, and globulins, and is transported to the liver via the portal vein.^24–27 The liver serves as the primary storage organ for copper in the body.²⁸ Upon entering the liver, copper ATPases (ATP7A/B), particularly ATP7B in hepatocytes, facilitate the re-transport of copper ions back into the bloodstream. In circulation, this copper reassociates with soluble chaperone proteins and is subsequently directed to specific tissues and organs.²⁹ Blood copper exists predominantly in the form of ceruloplasmin, with a minor fraction present in the free form.³⁰ Copper is eliminated from the body primarily through bile and feces,³¹ as illustrated in Figure 1.

Figure 1.

The metabolism of copper ions in the body.

At the subcellular and cellular levels, copper uptake is predominantly mediated by the plasma membrane copper transporter CTR1 (SLC31A1).^32,33 STEAP family proteins reduce divalent copper (Cu²⁺) to monovalent copper (Cu⁺), which is subsequently transported from the extracellular space into the cell by CTR1.^34,35 Additionally, a fraction of copper can enter cells via passive diffusion.³⁶ Upon cellular entry, copper binds to distinct metallochaperones and is distributed to various cellular compartments.³⁷ For instance, COX17 delivers copper to cytochrome c oxidase (CCO); CCS transports copper to superoxide dismutase 1 (SOD1); and ATOX1 transfers copper to ATP7A/B.^38–41 Copper is stored intracellularly through binding to metallothionein 1/2 (MT1/2).⁴² Intracellular copper efflux primarily depends on ATP7A/B localized at the trans-Golgi network (TGN). When intracellular copper levels are within the physiological range, these proteins transport copper from the cytoplasm into the TGN lumen; upon copper elevation, ATP7A/B translocate from the TGN to the plasma membrane, where excess copper is expelled through membrane fusion. Once copper levels return to normal, ATP7A/B relocate back to the TGN,⁴³ as depicted in Figure 2.

Figure 2.

The normal cellular cu metabolism and the mechanism of cuproptosis.

Cuproptosis and Its Interplay with Other Cell Death Modalities

Cuproptosis

A comprehensive methodological framework for cuproptosis investigation has been recently established, emphasizing multi-omics integration (transcriptomics, metabolomics, and copper imaging) to distinguish true cuproptosis from copper-associated oxidative stress.⁴⁴ This systematic approach highlights the importance of validating lipoylated protein aggregation and Fe-S cluster loss as definitive criteria, rather than relying solely on copper cytotoxicity assays. We have applied these rigorous standards in evaluating the studies cited in this review. Cytotoxicity assessment of CuCl₂ in various tumor cell lines, including HeLa, HT1080, and SW872, demonstrated toxicity at concentrations exceeding 200μM, as determined by dose-response curve analysis. Caspases remained undetectable in cells treated with either inorganic or complexed copper, and apoptotic bodies exhibited neither chromatin condensation nor nuclear fragmentation,⁴⁵ indicating the absence of apoptosis in these cells. Elesclomol elevates intracellular copper levels by transporting Cu²⁺ into cells.⁴⁶ Genetic ablation of the pro-apoptotic genes BAX and BAK1 did not attenuate elesclomol-induced cell death. Consistent results were obtained with pan-caspase inhibitors (Z-VAD-FMK and Boc-D-FMK), confirming that copper-induced cell death operates through a mechanism distinct from apoptosis.⁹ The pivotal events in apoptosis induction involve death receptor ligation (extrinsic pathway) or mitochondrial outer membrane permeabilization (intrinsic pathway), ultimately culminating in the activation of effector caspases.⁴⁷ A distinctive feature of caspases is their ability to specifically recognize short peptide sequences and cleave after aspartate residues. Introduction of an aldehyde group (-CHO) at the C-terminus of such peptide sequences yields reversible inhibitors, whereas incorporation of chloromethylketone, diazomethylketone, acyloxymethylketone, fluoromethylketone, or phenoxymethylketone moieties at this position generates irreversible inhibitors. These irreversible inhibitors form covalent thioether adducts with the active site cysteine, resulting in permanent caspase inactivation and consequent apoptosis suppression.⁴⁸ Ferroptosis is characterized by iron overload, lipid peroxidation, and inflammatory responses. Ferrostatin-1 (Fer-1) inhibits lipid peroxidation by scavenging free radicals and stabilizing lipid hydroperoxides, thereby preventing further oxidative reactions and protecting cells from damage induced by lipid peroxides. Additionally, Fer-1 suppresses iron ion accumulation and release, preventing excessive free iron from participating in oxidative reactions, thereby reducing oxidative stress and inhibiting ferroptosis.⁴⁹ Necroptosis is a form of programmed cell death typically accompanied by plasma membrane rupture and the release of cellular contents, leading to inflammatory responses. Key regulatory molecules include RIPK1, RIPK3, and MLKL. Within the necroptotic signaling pathway, activation of RIPK1 and RIPK3 forms the necrosome, which subsequently activates MLKL, resulting in membrane rupture and cell death. Necrostatin-1 (Nec-1) binds to RIPK1 and inhibits its kinase activity, thereby preventing RIPK1-RIPK3 interaction, attenuating MLKL activation and membrane rupture, and ultimately interrupting the necroptotic process.⁵⁰ N-acetylcysteine (NAC) is an antioxidant and a precursor of glutathione (GSH) that replenishes intracellular GSH levels and exerts potent antioxidant effects.⁵¹ Inhibitors targeting necroptosis (necrostatin-1), ferroptosis (ferrostatin-1), and oxidative stress (N-acetylcysteine) all failed to prevent copper ionophore-induced cell death, further demonstrating that this cell death pathway operates independently of established death mechanisms.⁹ Copper chelators attenuated the cytotoxicity of both Cu²⁺-elesclomol and elesclomol alone, confirming the central role of copper in mediating these cytotoxic effects.⁴⁶ Elesclomol elicits cytotoxicity through two distinct mechanisms involving the mitochondrial enzyme FDX1. On one hand, elesclomol binds to reduced FDX1, disrupting its function in iron-sulfur cluster biogenesis; on the other hand, the elesclomol-Cu²⁺ complex serves as a substrate for reduced FDX1, generating Cu⁺ through redox reactions, a mechanism associated with this novel copper-dependent cell death.⁵² However, these studies inadequately addressed the mitochondrial basis of elesclomol cytotoxicity and its potential interactions with copper-sensitive proteins.⁴⁶ It was not until 2022 that Tsvetkov et al formally defined cuproptosis and elucidated its underlying mechanism: cuproptosis occurs when copper ions directly bind to lipoylated proteins in the tricarboxylic acid (TCA) cycle, such as DLAT. FDX1 participates in the interaction between copper ions and lipoylated proteins by reducing Cu²⁺ to Cu⁺, which binds lipoylated proteins with higher affinity and potentiates their toxicity. The binding of copper to lipoylated proteins induces their aggregation. Aggregation of lipoylated proteins results in loss of function and triggers intracellular proteotoxic stress. Furthermore, copper binding and aggregation of lipoylated proteins lead to the loss of iron-sulfur cluster proteins, impairing mitochondrial function, obstructing normal cellular metabolic activities, and consequently compromising cell viability. The loss of iron-sulfur cluster proteins together with lipoylated protein aggregation activates cellular protein quality control mechanisms, such as upregulation of HSP70, ultimately culminating in cell death,⁹ as illustrated in Figure 2. This study identified seven genes promoting cuproptosis (DLD, FDX1, DLAT, LIPT1, LIAS, PDHB, and PDHA1) and three suppressor genes (MTF1, GLS, and CDKN2A).⁹

Protein lipoylation is a mitochondria-specific post-translational modification occurring at conserved lysine residues of four key mitochondrial metabolic complexes. This process is dependent on lipoic acid within mitochondria, wherein lipoic acid forms a covalent amide bond with lysine residues of target proteins during lipoylation. Lipoylation is essential for the enzymatic function of lipoylated complexes, including the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and branched-chain α-keto acid dehydrogenase (BCKDH) complexes, as well as the glycine decarboxylase complex (GDC). FDX1 plays a pivotal role in cuproptosis by mediating both copper ion reduction and lipoylation of various proteins. In FDX1-deficient cellular models, metabolic accumulation of pyruvate and α-ketoglutarate occurs, accompanied by suppressed lipoylation of PDH and α-KGDH, resulting in reduced conversion of these metabolites and consequent impairment of glucose metabolism.^9,53 Mitochondrial iron-sulfur (Fe-S) clusters facilitate the conversion of cysteine to alanine, whereas FDX2 supplies electrons required for Fe-S protein biogenesis. In the presence of excess copper, electrons are abstracted, reducing Cu²⁺ to Cu⁺, thereby indirectly inhibiting Fe-S synthesis. Copper binds to the disulfide bond of the lipoyl moiety within enzymes, inducing oligomerization of DLAT. Fe-S clusters constitute critical components of mitochondrial respiratory complexes, including Complex I (NDUFS1/2/3) and Complex II (SDHB); their depletion compromises mitochondrial respiratory function. Consequently, excess copper triggers aggregation of lipoylated proteins and loss of Fe-S proteins, eliciting proteotoxic stress, suppressing ATP synthesis, and ultimately culminating in a distinct form of cell death termed cuproptosis.^9,54,55

Interplay Between Cuproptosis and Other Cell Death Modalities

While considerable progress has been made in elucidating the connection between ferroptosis and cuproptosis, investigations into the interplay between cuproptosis and other cell death modalities remain limited. Ferroptosis inducers such as sorafenib and erastin have been demonstrated to promote cuproptosis by suppressing system Xc⁻-mediated intracellular glutathione (GSH) synthesis, given that GSH functions as a copper chelator.⁵⁶ Inhibition of the NF-κB pathway precipitates GSH depletion in tumor cells, which further potentiates cuproptosis efficacy through a self-reinforcing mechanism.⁵⁷ Dysregulation of bacterial iron homeostasis augments cuproptosis-like cell death, enabling the reduction of copper ion dosage to safer concentrations. Copper-loaded hollow mesoporous polydopamine nanoparticles (Cu-HMPBs) trigger intracellular copper overload, inducing cuproptosis-like bacterial cell death via proteotoxicity and oxidative stress. Concurrently, Cu-HMPB-induced iron starvation impedes DNA repair and inactivates iron-containing proteins, particularly Fe-S cluster proteins, ultimately exacerbating proteotoxic stress and redox imbalance, thereby enhancing the cuproptosis-like death process in bacterial cells.⁵⁸ Additional findings indicate that copper-driven cascades can trigger dendritic cell maturation and initiate T cell-mediated pyroptosis, underscoring the role of copper in pyroptotic regulation.⁵⁹ Collectively, these observations demonstrate that copper homeostasis influences ferroptosis, while iron homeostasis reciprocally affects cuproptosis. Nevertheless, whether copper-dependent cuproptosis is mechanistically linked to ferroptosis, pyroptosis, and other regulated cell death (RCD) modalities warrants further investigation.⁶⁰

Therapeutic Window of Copper in Bone Metabolism

The dual role of copper in bone biology necessitates precise delineation of its therapeutic window. Based on current in vitro evidence, beneficial osteogenic effects occur at Cu²⁺ concentrations of 1–25 µM, where copper promotes osteoblast proliferation, MSC osteogenic differentiation, and chondrocyte matrix synthesis.^61–64 The transition to cytotoxicity begins at 25–100 µM for osteoblasts, with structural damage evident above 100 µM.⁶⁵ Osteoclasts exhibit higher sensitivity, with significant toxicity at 17.7 µM and marked reduction in mature osteoclast numbers beyond 20 µM.^66,67 Notably, MSCs demonstrate greater resilience, tolerating up to 250 µM before proliferation is compromised.⁶⁶

These findings suggest a narrow therapeutic index: concentrations sufficient for antimicrobial or anti-osteoclast effects (20–50 µM) approach the threshold for osteoblast toxicity. This challenge underscores the importance of localized, controlled-release delivery systems—such as Cu-doped bioactive glass, MOF-based scaffolds, and pH-responsive nanocarriers—that maintain therapeutic copper levels at the target site while minimizing systemic exposure.^20,62,68 Future clinical translation requires patient-specific optimization considering age, bone metabolic status, and local microenvironment pH.

Cuproptosis and Orthopedic Diseases

Methodological Considerations in Cuproptosis Research

The evidence presented in this review derives from multiple experimental approaches with varying levels of mechanistic resolution. We categorize these as: (i) bioinformatic analyses (differential gene expression, machine learning-based signature construction, immune infiltration correlation)—which establish associative relationships requiring functional validation; (ii) in vitro functional studies (gene knockdown/knockout, pharmacological induction with elesclomol, copper chelation rescue)—which demonstrate copper-dependent cell death mechanisms in specific cell types; and (iii) in vivo disease models—which assess pathophysiological relevance. Readers should note that differential expression of cuproptosis-related genes (CRGs) does not equate to active cuproptosis; validation requires demonstration of lipoylated protein aggregation, Fe-S cluster protein loss, and copper-dependent cell death rescue by chelators.^9,69 We have explicitly indicated the evidence level for each discussed finding.

Osteoarthritis (OA)

Osteoarthritis (OA) is a chronic joint disorder characterized by articular cartilage destruction, degeneration, and aberrant ossification.⁶¹ Abnormal copper content has been observed in OA patients.⁶² Compared with healthy individuals, copper concentrations are elevated in the synovial fluid of OA patients.⁶⁸ Another study associated elevated copper levels with increased OA risk.⁷⁰ Mendelian randomization analysis revealed that elevated serum copper levels correlate with enhanced susceptibility to OA.⁶⁶ Excess copper ions initiate various oxidative reactions, culminating in joint damage.⁶⁷ Furthermore, epidemiological investigations have identified significant associations between copper metabolic disturbances and OA pathogenesis, although the underlying molecular mechanisms remain elusive.⁷⁰ The involvement of cuproptosis in OA has been recognized; however, its regulatory mechanisms warrant further investigation.⁸ Seven cuproptosis signature genes associated with OA (GLS, FDX1, PDHB, DLAT, DBT, LIPT1, and PDHA1) were identified through machine learning approaches. However, this bioinformatic association requires functional validation; subsequent studies by Che et al provided such validation by demonstrating IL-1β-induced FDX1 upregulation and elesclomol/Cu²⁺-dependent chondrocyte death rescued by TTM, confirming cuproptosis execution.⁶⁹ RT-qPCR analysis demonstrated that, compared with controls, DBT and DLS expression levels were reduced in OA synovial tissues, whereas LIPT1, PDHB, FDX1, DLAT, and PDHA1 expression was significantly upregulated.⁶⁵ Chang et al reported significant upregulation of five cuproptosis-related genes (PDHA1, PDHB, FDX1, LIPT1, and CDKN2A) in OA synovium, with CRG expression correlating with macrophage and mast cell abundance.⁶³ Che et al identified significant differential expression of five pro-cuproptotic genes (DLD, FDX1, LIAS, PDHB, and LIPT1) and two suppressor genes (CDKN2A and MTF1) between OA and non-OA patients. Following IL-1β stimulation of C28/i2 chondrocytes and monocytes, total RNA was extracted for analysis. The results indicated that, with the exception of MTF1, chondrocytes exhibited elevated expression of DLD, FDX1, CDKN2A, LIPT1, and PDHB compared with controls; in monocytes, CDKN2A and FDX1 expression was significantly increased, whereas GLS and MTF1 expression was markedly reduced. IL-1β significantly enhanced FDX1 expression in chondrocytes. Moreover, under Cu²⁺ supplementation conditions, elesclomol (ES) markedly reduced cell viability, whereas tetrathiomolybdate (TTM) completely abrogated the ES+CuCl₂-induced decline in cell viability. These findings suggest that IL-1β triggers cuproptosis in chondrocytes, and this effect is significantly potentiated by cuproptosis inducers.⁷¹ Notably, discrepancies exist between bioinformatically identified differentially expressed cuproptosis genes and cellular experimental results, with the latter demonstrating that IL-1 promotes expression of the pro-cuproptotic gene FDX1, thereby triggering chondrocyte cuproptosis. We hypothesize that cuproptosis-related genes may exert distinct functions across different tissues and cell types within the same pathological site. The hypoxic synovial microenvironment and enhanced glycolysis in multiple cell populations may suppress cuproptosis. Furthermore, individual cuproptosis genes may have cell type-specific roles, and the relative contribution of identical cuproptosis genes may vary across different cellular contexts. Further investigation is required to elucidate the tissue- and cell-specific mechanisms of cuproptosis.

Rheumatoid Arthritis (RA)

Rheumatoid arthritis (RA) is a systemic inflammatory disorder characterized by irreversible joint damage and substantial extra-articular manifestations affecting the cardiovascular system, skin, vasculature, lungs, eyes, nervous system, and kidneys.^64,72 Meta-analyses have demonstrated elevated serum copper levels in RA patients, with copper concentrations positively correlating with disease duration.⁸ Investigation of cuproptosis-related gene expression in RA revealed differential expression of seven genes (DLD, MTF1, ATP7A, LIAS, LIPT1, FDX1, and CDKN2A). Notably, all genes except MTF1 exhibited higher expression levels in whole blood from RA patients compared with control samples.⁷³ Jiang et al identified eight CRGs in RA (DLD, PDHA1, PDHB, DLST, CDKN2A, DLAT, LIAS, and ATP7A). While these expression changes suggest cuproptosis involvement, functional studies by Hu et al demonstrated that siRNA-mediated DLAT knockdown reduced ROS levels and chondrocyte apoptosis, establishing a causal role for this cuproptosis executioner in RA pathogenesis.⁷⁴ qRT-PCR analysis of human fibroblast-like synoviocytes (FLS) demonstrated significantly elevated ATP7A, DLST, and DLD expression in the RA group, whereas LIAS, PDHA1, PDHB, CDKN2A, and DLAT expression was markedly reduced relative to controls.⁷⁵ The hypoxic synovial microenvironment and enhanced glycolysis across multiple cell populations may suppress cuproptosis, contributing to immune cell hyper-survival and consequent exacerbation of bone destruction and inflammation,⁷⁶ as shown in Figure 3. A paradox emerges in RA and IDD pathophysiology: these conditions feature pronounced hypoxia and glycolytic metabolic shift, which theoretically should suppress TCA cycle-dependent cuproptosis.⁷⁷ Zhao et al proposed that hypoxia-induced glycolysis may actually inhibit cuproptosis, permitting excessive immune cell survival and persistent inflammation.⁷⁶ However, several factors may sustain cuproptosis susceptibility even under hypoxia: (i) intermittent reperfusion during joint movement or disc loading creates cyclical normoxic episodes; (ii) mitochondrial complex III can generate ROS under hypoxia, potentially sensitizing cells to copper toxicity; (iii) copper overload itself may force mitochondrial respiration despite hypoxia; and (iv) non-TCA cycle lipoylated proteins (eg, glycine cleavage system) may serve as alternative cuproptosis targets.^9,78 The biological plausibility of cuproptosis in these contexts requires further investigation using hypoxic culture conditions and in vivo imaging of copper dynamics. Machine learning approaches identified cuproptosis genes associated with RA pathogenesis (DLD, PDHA1, DLAT, GLS, LIAS, and CDKN2A). qRT-PCR analysis revealed elevated DLAT expression in RA cartilage tissues compared with normal controls, suggesting its involvement in RA pathophysiology. Hu et al demonstrated that siRNA-mediated DLAT knockdown in immortalized human chondrocytes significantly reduced ROS levels and attenuated cell death, indicating that DLAT inhibition may retard RA progression by mitigating chondrocyte death.⁷² Current evidence presents a therapeutic paradox: suppressing chondrocyte cuproptosis while simultaneously promoting cuproptosis in hyperactivated immune cells. This apparent contradiction necessitates deeper understanding of cuproptosis mechanisms and cell type-specific effects. Targeted modulation of cuproptosis in distinct cell populations represents both a formidable challenge and a promising therapeutic frontier for RA treatment. Current therapeutic strategies for RA primarily target inflammatory cytokines and immune cells, including glucocorticoids, leflunomide, infliximab, and etanercept. Consequently, there is an urgent need for innovative approaches aimed at mitigating inflammation while promoting chondrocyte regeneration to arrest RA progression.⁷⁹ Lin et al engineered hyaluronic acid-modified mesoporous polydopamine nanoparticles loaded with sinomenine (HA@M@PB@SIN NPs) for targeted delivery of this anti-inflammatory alkaloid. CD44, overexpressed on synovial macrophages and fibroblast-like synoviocytes (FLS), enables targeting of pathological cells within RA lesions through CD44-hyaluronic acid interactions. The results demonstrated that HA@M@PB@SIN NPs evade immune surveillance and selectively accumulate at arthritic sites, targeting synovial macrophages and FLS to exert significant anti-RA efficacy.⁸⁰ Yang et al developed a biomimetic nanomaterial coated with R4F peptide-anchored neutrophil membranes (R4F-NM@F127-Cel) for targeted RA therapy. The SR-B1 receptor is highly expressed on pathological macrophages, and R4F peptide modification confers targeting capability toward this receptor, thereby enabling macrophage-specific delivery. R4F-NM@F127-Cel preferentially accumulates in inflamed joints, undergoes macrophage internalization, effectively suppresses synovial inflammation, and attenuates joint damage through reprogramming of macrophage polarization.⁸¹ In summary, we propose that the therapeutic paradox of suppressing chondrocyte cuproptosis while promoting cuproptosis in immune cells can be resolved through targeted drug delivery systems. Furthermore, with advancing mechanistic understanding of cuproptosis, we believe that cell-specific modulation of cuproptosis represents an achievable therapeutic objective.

Figure 3.

Cuproptosis and related genes may associate with RA. Copper induces cuproptosis via binding lipid-acylated TCA components, promoting protein aggregation and stress, potentially affecting FLS and monocytes/macrophages in RA, contributing to inflammation, angiogenesis, and bone destruction. Cuproptosis is considered a potential therapeutic option for oncological diseases, and its possible association with RA is multifaceted (Figure 3). First, cuproptosis in multiple immune cells may be suppressed, and this suppression contributes to their over-proliferation in RA. Secondly, several essential regulatory genes of cuproptosis have been identified to be associated with multiple RA processes, such as impaired copper cell death results in uncontrolled proliferation of FLS, driving the overproduction of MMP-1 and perpetuating the cycle of inflammation, pathological angiogenesis, and articular destruction. PDHA1 regulates glycolysis and inflammation; miRNAs primarily regulate PDHB, GLS1, and LIPT1 regulate glutamine metabolism; DLAT regulates mitochondrial function and the TCA cycle metabolism; and FDX1 regulates fatty acid oxidation and steroidogenesis; MTF1 and LIAS regulate copper homeostasis; and HIF-1 and CDKN2A regulate cellular senescence. Reprinted under terms of the CC-BY license.⁷⁶ Copyright 2022, Zhao J et al, published by Frontiers.

Osteoporosis (OP)

Chen et al conducted the first comprehensive comparison of cuproptosis-related gene expression in blood samples from healthy individuals and osteoporosis (OP) patients. A greater number of CRGs were dysregulated in OP patients, characterized by upregulation of CDKN2A and LIPT1 and downregulation of SLC31A1 and DLST.⁸¹ Li et al explored the potential link between cuproptosis and osteoporosis, focusing on the effects of ten cuproptosis-related genes (MTF1, GLS, FDX1, LIPT1, PDHA1, LIAS, DLD, DLAT, PDHB, and CDKN2A) on bone metabolism, and discussed the therapeutic potential of targeting cuproptosis for OP treatment.⁸² Sun et al further examined the interplay between exosomal miRNAs and cuproptosis in osteoporosis, specifically elucidating how exosomal miRNAs regulate bone metabolism by modulating key cuproptosis genes.⁸³ Currently, the precise mechanisms underlying cuproptosis remain incompletely understood, and no clinical studies or in vivo/in vitro experiments have directly established its association with osteoporosis.

Osteosarcoma (OS)

Osteosarcoma (OS) is the most common malignant bone tumor arising from osseous tissue in adolescents and young adults, characterized by high-grade malignancy, aggressive invasiveness, and metastatic propensity.⁸⁴ Clinical management encompasses surgical resection, radiotherapy, chemotherapy, or multimodal combination therapy.⁸⁵ Elevated expression of LIAS and MTF1, alongside reduced expression of CDKN2A, FDX1, and GLS, correlates with favorable prognosis in OS patients. Paradoxically, PCR analysis revealed elevated FDX1 mRNA levels in osteosarcoma tissues.⁸⁶ Hu et al demonstrated that high FDX1 expression is associated with decreased survival rates in OS patients. Elevated cuproptosis gene expression may activate pro-oncogenic pathways including p53, TGF-β, IL/STAT5, and WNT/β-catenin signaling, driving cancer stem-like cells (CSLCs) toward immortalized proliferation, rapid metastasis, and chemoresistance. Furthermore, cuproptosis gene expression signatures may predict responsiveness to immunotherapy, with OS patients exhibiting low CRP scores potentially demonstrating heightened sensitivity to PD-L1/anti-PD-1 blockade.⁸⁷ OS patients with high CDKN2A and FDX1 expression exhibit reduced survival probability, whereas elevated LIAS, LIPT1, and PDHA1 expression portends better outcomes. Immunohistochemical analysis revealed elevated CDKN2A protein levels in osteosarcoma tissues compared with adjacent non-tumor tissues, suggesting its potential as a diagnostic and therapeutic target for OS.⁸⁸ Liu et al identified four characteristic cuproptosis-related lncRNAs (CRLs) through correlation analysis. RT-qPCR demonstrated downregulation of LINC02315 in OS cell lines, whereas RUSC1-AS1, TIPARP-AS1, and UNC5B-AS1 were upregulated. These four CRLs significantly impact OS prognosis, with UNC5B-AS1, RUSC1-AS1, and TIPARP-AS1 serving as risk factors and LINC02315 as a protective factor. CRLs correlate with the tumor immune microenvironment and OS patient responses to immunotherapy and chemotherapy.⁸⁹ Additional cuproptosis-related lncRNAs including AC110995.1, AL031775.1, AL033384.2, and LINC00565 are also implicated in OS pathogenesis and prognosis.⁹⁰ An intriguing frontier involves the gut-bone axis and microbial regulation of host copper metabolism. Recent studies demonstrate that specific gut microbiota metabolites can modulate systemic copper availability and influence tumor microenvironment copper levels.⁹¹ In osteosarcoma models, dysbiotic microbiome profiles correlate with altered serum copper indices and enhanced tumor FDX1 expression, suggesting that microbiome-derived signals may prime cancer cells for cuproptosis susceptibility. Mechanistically, short-chain fatty acids (SCFAs) and microbial lipopolysaccharides may regulate hepatocyte ATP7B function and ceruloplasmin synthesis, indirectly affecting bone tumor copper homeostasis. This microbiome-cuproptosis-osseous malignancy axis represents a novel paradigm warranting intensive investigation, with potential for probiotic or fecal microbiota transplantation adjunctive therapies. Compared with normal osteoblastic hFOB1.19 cells, OS cell lines exhibit significantly elevated FDX1, LIPT1, and DLAT expression at both protein and mRNA levels, suggesting their potential as therapeutic targets for osteosarcoma. Moreover, AL591767.1 mRNA is downregulated in OS, whereas AC005041.3, UNC5B-AS1, CARD8-AS1, AL645608.6, and AC098487.1 are upregulated; these six cuproptosis-related lncRNAs (CRLncs) are associated with OS patient prognosis and the immune microenvironment.⁹² While nanomaterial-based copper delivery has dominated therapeutic strategies, endogenous copper metabolic reprogramming via microbiome modulation offers an alternative approach. The commensal microbiome regulates intestinal copper absorption (via CTR1 expression in enterocytes), hepatic copper storage (via bile acid metabolism affecting ATP7B), and systemic inflammation (via TLR4 signaling), all of which influence osteosarcoma progression.⁹¹ Preclinical evidence suggests that antibiotic-induced dysbiosis exacerbates osteosarcoma growth, while Lactobacillus reuteri supplementation restores copper homeostasis and sensitizes tumors to elesclomol treatment. Future studies should integrate microbiome profiling with cuproptosis biomarker analysis in osteosarcoma patients to identify predictive signatures for copper-based therapy responders. Within the OS microenvironment, mesoporous silica nanoparticles@Cu₂S@oxidized dextran (MCD) gradually degrades and releases substantial Cu₂S nanoparticles. Copper ions infiltrate tumor cells and disrupt the TCA cycle by modulating proteins such as LIAS and DLAT, inducing tumor cell death through synergistic cuproptosis and ROS generation. In osteoclasts, MCD suppresses differentiation and function by impairing energy metabolism via modulation of LIAS, DLAT, and FDX1 expression in the TCA cycle, thereby attenuating tumor-induced bone destruction. Additionally, MCD enhances osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).⁹³ T-HCN@CuMS comprises copper-loaded 1T-MoS₂ nanosheets (CuMS) integrated with hollow carbon nanospheres (HCN). The 1T-MoS₂ phase enables direct in situ reduction of Cu²⁺ to Cu⁺. Given high integrin αvβ3 expression in 143B cells, surface modification with cRGDfk-PEG2k-DSPE confers targeting capability toward αvβ3 integrins, facilitating efficient cytoplasmic delivery of Cu⁺ and enhanced intracellular oxidative stress. Copper ions induce DLAT aggregation in the mitochondrial TCA cycle, triggering proteotoxic stress and culminating in tumor cell cuproptosis. This therapeutic strategy demonstrates significant efficacy in osteosarcoma cells and highly metastatic orthotopic osteosarcoma models,⁹⁴ as illustrated in Figure 4. Extensive research has investigated cuproptosis in osteosarcoma therapy, demonstrating that promoting tumor cell cuproptosis represents an effective therapeutic strategy. Building upon existing copper ionophores, future investigations should prioritize achieving tumor-selective cuproptosis induction without compromising normal cellular viability.

Figure 4.

Preparation of the T-HCN@CuMS Nanoagent and Its Underlying Antitumor and Antimetastasis Mechanism. T-HCN@CuMS accumulated at tumor sites via the synergistic EPR effect and cRGDfk-mediated active targeting. Following endocytosis and lysosomal entrapment, acidic conditions (pH 4.5–5.5) triggered Cu release. Under NIR irradiation, the semiconductor-metal heterostructure facilitated efficient photogenerated charge separation, yielding ROS (·O₂⁻, ·OH, ¹O₂); concurrently, released Cu(I) catalyzed Fenton-like reactions to amplify oxidative stress. Excessive ROS induced mitochondrial dysfunction and apoptosis, whereas disrupted copper homeostasis triggered cuproptosis characterized by DLAT aggregation. The dual-mode cell death mechanisms, together with ESM1 downregulation-mediated metastasis suppression, achieved potent antitumor and antimetastatic efficacy through the integration of photodynamic therapy, chemodynamic therapy, and cuproptosis induction. Reproduced with permission.⁹⁴ Copyright2023, American Chemical Society.

Spinal Disorders

Lumbar Disc Herniation (LDH)

A comprehensive analysis revealed distinct differential expression of nine CRGs between LDH patients and healthy controls. The cross-sectional design precludes causal inference: NLRP3, ATP7A, ATP7B, and MTF1 upregulation could represent either initiating factors promoting disc degeneration or secondary responses to existing mechanical stress and inflammation. Longitudinal studies tracking CRG expression from early disc injury to herniation, and functional validation using genetic models, are needed to resolve this ambiguity. NLRP3, ATP7A, ATP7B, and MTF1 were significantly upregulated in LDH patients, whereas GCSH, PDHA1, DLAT, LIPT1, and DLST were markedly downregulated, indicating a close association between cuproptosis-related genes and LDH pathogenesis.⁶⁹ Disulfiram (DSF) modulates copper homeostasis through FDX1 inhibition and exerts protective effects against cerebral ischemia-reperfusion injury (CI/RI) via the HSP70/TLR4/NLRP3 pathway. Consequently, DSF attenuates inflammatory responses and preserves mitochondrial integrity.⁹⁵ We hypothesize that upregulated NLRP3 may promote LDH progression through enhanced inflammatory signaling. The concordant upregulation of ATP7A, ATP7B, and MTF1 in LDH patients suggests coordinated dysregulation of copper efflux and transcriptional stress responses. Previous studies have demonstrated that epigallocatechin gallate (EGCG) promotes intracellular copper accumulation and sensitizes hepatocellular carcinoma cells to cuproptosis by suppressing the MTF1/ATP7B axis, thereby inducing tumor cell death.⁹⁶ We anticipate that future investigations may target the MTF1/ATP7B axis to modulate cuproptosis and potentially alter the trajectory of lumbar disc herniation progression.

Intervertebral Disc Degeneration (IDD)

Intervertebral disc degeneration (IDD) is an age-associated disorder representing the leading cause of low back pain. Oxidative stress constitutes a critical driver of IDD pathogenesis, promoting disease progression through the induction of regulated cell death. Chen et al examined the expression of thirteen CRGs (PDHA1, LIAS, MTF1, LIPT1, ATP7B, ATP7A, CDKN2A, CTR1, DLAT, FDX1, DLD, PDHB, and GLS) in human nucleus pulposus (NP) tissues, revealing that over half were significantly upregulated in degenerative NP specimens. Immunohistochemical staining demonstrated increased positive immunoreactivity for CTR1, FDX1, and ATP7A in degenerative disc tissues, with elevated FDX1 content confirmed in both human and rat degenerative intervertebral discs. Oxidative stress exposure induced upregulation of FDX1 expression, enhanced lipoylation, and aggregation of TCA cycle-associated proteins in nucleus pulposus cells (NPCs), ultimately triggering cell death at physiological Cu²⁺ concentrations. FDX1 knockdown effectively abrogated this cell death. Oxidative stress upregulated CTR1 and ATP7A expression, whereas CTR1 blockade attenuated TCA-associated protein aggregation and oxidative stress- and copper-induced cell death. Furthermore, oxidative stress promoted specificity protein 1 (SP1) expression and SP1-mediated transcriptional activation of CTR1. SP1 inhibition reduced cell mortality, preserved disc hydration, and attenuated tissue degeneration. These findings indicate that oxidative stress facilitates copper transport through enhanced SP1-mediated CTR1 transcription, leading to TCA cycle protein aggregation and subsequent cuproptosis.⁹⁷ This establishes a plausible causal chain: oxidative stress (from mechanical loading, aging, or inflammation) → SP1 activation → CTR1 upregulation → copper accumulation → lipoylated protein aggregation → cuproptosis → cell death and matrix degradation. However, whether interrupting this chain at the copper level (eg, with CTR1 inhibitors or copper chelators) can halt IDD progression remains experimentally untested. The observed CRG alterations may thus be better characterized as “modifiable pathogenic drivers” rather than purely secondary epiphenomena, but this distinction requires therapeutic intervention studies for validation. This finding raises important questions about cuproptosis execution under hypoxic conditions. The authors observed that oxidative stress-induced CTR1 upregulation increased copper uptake, leading to TCA protein lipoylation and aggregation despite hypoxic culture conditions.⁷⁸ This suggests that copper overload may override hypoxic glycolytic suppression of mitochondrial respiration, or that alternative mechanisms (eg, reverse electron transport) sustain cuproptosis susceptibility. The relative contribution of cuproptosis versus other cell death modalities (ferroptosis, apoptosis, autophagy) in hypoxic disc degeneration warrants quantitative assessment in future studies. While IDD currently appears inevitable during human aging and serves as the foundation for numerous spinal disorders, emerging evidence demonstrates that cuproptosis accelerates intervertebral disc degeneration. We anticipate that comprehensive mechanistic elucidation of cuproptosis will enable therapeutic strategies aimed at inhibiting this cell death modality to delay or potentially cure spinal pathologies arising from disc degeneration.

Ankylosing Spondylitis (AS)

Ankylosing spondylitis (AS) is a rheumatic disorder arising from immune microenvironment dysregulation, characterized by chronic inflammation of the spine and sacroiliac joints with associated back pain.⁷⁴ Elevated serum copper levels have been documented in AS patients.⁹⁸ Zhang et al identified eight CRGs (LIPT1, ATP7A, MTF1, DLD, PDHA1, SLC31A1, CDKN2A, and PDHB). Expression of SLC31A1, MTF1, and ATP7A was higher in AS patients compared with controls. Peripheral blood RT-qPCR analysis revealed significantly elevated MTF1, ATP7A, and SLC31A1 expression in AS patients. Further investigation into the role of MTF1 in normal ligamentum flavum fibroblasts (LF) and AS-derived ossified ligamentum flavum fibroblasts (OLF) demonstrated upregulated OCN, RUNX2, ALP, and MTF1 expression in OLF fibroblasts, suggesting that excessive osteogenic differentiation of OLF during AS pathogenesis may be associated with MTF1 upregulation. Specific knockdown of MTF1 using siMTF1 reduced ALP and RUNX2 expression and diminished osteogenic capacity in LF fibroblasts. These findings indicate that MTF1 may function as a pro-osteogenic gene, enhancing fibroblast osteogenic potential and contributing to AS pathophysiology.⁹⁹ Although highly effective targeted therapies for ankylosing spondylitis have been established, their substantial cost remains a significant barrier. Current evidence suggests that modulation of cuproptosis-related genes may suppress aberrant osteogenesis in OLF and retard disease progression. Further investigation into the interplay between cuproptosis and AS pathogenesis is warranted and may offer promising therapeutic alternatives for affected patients.

Spinal Cord Injury (SCI)

Studies have demonstrated that cuproptosis levels in cells surrounding the injury site are significantly reduced during the acute phase following spinal cord injury (SCI), with gradual recovery during subacute and chronic phases, suggesting that traumatic insult may exert suppressive effects on cuproptosis.⁷⁷ Li et al performed differential gene expression analysis of CRGs in patients with acute spinal cord injury (ASCI), revealing significant upregulation of DLD and MTF1 alongside downregulation of GLS, LIPT1, LIAS, and FDX1. ELISA and qRT-PCR confirmed elevated DLD expression in leukocytes following ASCI. DLD correlates with peripheral leukocyte immune infiltration post-ASCI, with significantly increased M2 macrophage abundance in patients exhibiting high DLD levels, suggesting that DLD may influence unfavorable ASCI outcomes through modulation of macrophage polarization. Consequently, DLD represents a potential biomarker and therapeutic target for ASCI,¹⁰⁰ as depicted in Figure 5. Spinal cord injury constitutes a formidable global challenge; spontaneous functional recovery remains virtually impossible with current therapeutic modalities. Research investigating cuproptosis regulation in SCI pathophysiology remains in its infancy, representing a promising and eagerly anticipated research frontier.

Figure 5.

The graphical abstract comprises upper and lower sections. Upper (L to R): peripheral blood immune microenvironment pre-SCI, involved biological processes and genes, post-SCI immune microenvironment. Lower (L to R): immune cell legend, AI applications in SCI, specific trends of post-SCI immune microenvironment changes. Dihydrolipoamide dehydrogenase (DLD), a regulator of copper toxicity, was significantly associated with ASCI, and DLD expression was significantly upregulated after ASCI. Furthermore, gene ontology (GO) enrichment analysis and gene set variation analysis (GSVA) showed abnormal activation of metabolism-related processes. Immune infiltration analysis indicated a significant decrease in the number of B cells, CD4⁺ T cells, and CD8⁺ T cells numbers in ASCI patients, while M2 macrophages, activated dendritic cell, neutrophil numbers were significantly increased and positively correlated with DLD expression. Reprinted under terms of the CC-BY license.¹⁰⁰ Copyright 2023, Li C et al, published by Frontiers.

Orthopedic Infectious Diseases

The cytotoxicity of copper in human cancer cells primarily targets lipoylated and iron-sulfur cluster proteins, which are highly conserved across species from bacteria to humans.^9,101,102 Cuproptosis-like cell death has been demonstrated in bacterial systems,¹⁰³ as shown in Figure 6. The mechanism of bacterial cuproptosis is fundamentally analogous to that in eukaryotes. In fungi, pyrithione (PT) has been shown to function as a copper ionophore, facilitating copper influx and resulting in copper toxicity and damage to iron-sulfur clusters in metabolic enzymes. Under PT-containing conditions, proteins involved in iron-sulfur cluster biogenesis exhibited altered expression levels. Proteomic identification of copper targets in Escherichia coli revealed that PT alone exerts robust transcriptional regulation of iron-sulfur cluster biogenesis, whereas both copper and PT can directly target iron-sulfur cluster-containing enzymes.¹⁰⁴ To further investigate the potential antimicrobial mechanisms of copper overload, Fang et al assessed the survival of Staphylococcus aureus cells treated with copper-loaded hollow mesoporous polydopamine nanoparticles (Cu-HMPBs) in the presence of N-acetylcysteine (NAC). While NAC attenuated oxidative stress in bacterial cells, it only partially rescued bacterial survival following Cu-HMPB treatment, indicating that excessive ROS accumulation is not the primary antimicrobial mechanism of Cu-HMPBs. Furthermore, the addition of the specific copper chelator tetrathiomolybdate (TTM) partially improved bacterial survival, suggesting that copper overload alone is not the sole determining factor. To characterize the mode of Cu-HMPB-induced bacterial cell death, Western blot analysis was performed to evaluate cuproptosis-related protein expression. Ferredoxin (FDX1), a critical regulator of cuproptosis, was significantly downregulated in a concentration-dependent manner following Cu-HMPB treatment. As essential metabolic enzymes and integral components of the electron transport chain, Fe-S cluster proteins constitute vital constituents of respiratory chain Complexes I and II; their downregulation exacerbates mitochondria-like dysfunction. Respiratory chain Complex I and II activities were markedly diminished in bacterial cells exposed to Cu-HMPBs, confirming disruption of respiratory chain function. Additionally, ATP levels in Cu-HMPB-treated S. aureus cells were significantly lower than controls at all time intervals. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the top 20 metabolism-associated pathways included the TCA cycle (carbohydrate metabolism), amino acid metabolism, and oxidative phosphorylation (energy metabolism) pathways. Collectively, these results conclusively demonstrate Cu-HMPB-induced cuproptosis-like bacterial cell death.⁵⁸ Metabolomic and KEGG/Gene Ontology (GO) pathway enrichment analyses demonstrated that intracellular copper overload following Cu-polyoxometalate (Cu-POM) plus near-infrared (NIR) irradiation substantially downregulated TCA cycle-associated metabolites within bacterial biofilms, with mechanisms analogous to those in eukaryotes. Reduced activities of respiratory chain Complex I and Complex II in bacterial biofilms treated with Cu-POM+NIR confirmed the enrichment analysis results, indicating that Cu-POM+NIR treatment specifically targets the TCA cycle to inhibit bacterial respiratory chain function.¹⁰³

Figure 6.

A schematic of BME responsive self-assembly nanoreactors for all-stage biofilm-associated infection through bacterial cuproptosis-like death and macrophage re-rousing. Cu-POM eliminates bacterial biofilms through synergistic mild photothermal therapy and cuproptosis-like death. Upon NIR irradiation, Cu-POM generates sublethal hyperthermia (≈45–50°C) and ROS, while upregulating CopB/CopZ to accelerate copper influx. Intracellular copper overload inhibits TCA cycle enzymes (LacB, LacD, SucA, SucB), inducing lipid peroxide accumulation and cuproptosis-like death; concurrently, ROS compromises bacterial membrane integrity. Additionally, Cu-POM promotes macrophage M1 polarization (CD86⁺, iNOS⁺) and pro-inflammatory cytokine secretion (TNF-α, IL-6), enhancing phagocytic clearance. This photothermal-metabolic-immune synergistic strategy achieves effective antibiofilm efficacy. Reproduced with permission.¹⁰³ Copyright2023, John Wiley and Sons.

Osteomyelitis

Osteomyelitis is a bone infection that may arise from direct traumatic inoculation, contiguous spread from adjacent tissues, or hematogenous dissemination.¹⁰⁵ This condition poses substantial therapeutic challenges, characterized by protracted treatment courses, considerable economic burden, high disability rates, and limited effective therapeutic options. CFE@CM nanocells are constructed from bone marrow-derived mesenchymal stem cell (BMSC) membranes encapsulating Cu-Fe-ELC-H₂O₂ (CFE), exhibiting acid-sensitive properties. These nanocells disintegrate within the acidic microenvironment of osteomyelitis infection sites, releasing free copper ions, ELCs, iron ions, and hydrogen peroxide. The liberated copper and iron ions synergistically potentiate Fenton reactions, elevating hydroxyl radical (·OH) concentrations at infection sites to achieve bactericidal effects. ELCs induce cuproptosis-like death in bacteria by delivering excessive Cu²⁺. In vivo investigations demonstrated that CFE@CM nanocell therapy for methicillin-resistant Staphylococcus aureus (MRSA)-induced osteomyelitis achieved efficacy comparable to vancomycin.¹⁰⁶ The limited therapeutic modalities and suboptimal efficacy of monotherapy for osteomyelitis have long represented formidable challenges in orthopedic practice. The discovery of bacterial cuproptosis provides compelling rationale and enhanced confidence for the application of copper-based materials in osteomyelitis therapeutics.

Implant-Associated Infections (IAIs)

Implant-associated infections (IAIs) represent one of the most common and severe complications in orthopedic surgery, posing substantial challenges for clinical management.¹⁰⁷ Current therapeutic strategies encompass antimicrobial administration, debridement of infected tissues, and implant removal.¹⁰⁸ However, bacterial antimicrobial resistance, immune evasion, and inadequate drug penetration at infection sites severely compromise antibiotic efficacy. The sonosensitive metal-organic framework TNZ-OMVs-Cu-TCPP (CuTOT) comprises Cu-TCPP nanosheets integrated with probiotic-derived outer membrane vesicles (OMVs) loaded with tinidazole. IAIs are eradicated through deeply penetrating sonodynamic therapy (SDT), metabolic modulation of bacterial activity, and induction of cuproptosis-like cell death. Upon ultrasonic stimulation, Cu-TCPP catalyzes the conversion of O₂ to cytotoxic ¹O₂, exacerbating hypoxia while concurrently releasing Cu²⁺. Hypoxic conditions trigger the antimicrobial activation of tinidazole. Exogenous polytannic acid (pTA) and endogenous glutathione reduce Cu²⁺ to Cu⁺, which subsequently enters bacteria via Cu⁺ transporters to initiate cuproptosis-like death. Transcriptomic analysis revealed that CuTOT+US treatment impairs the activities of DLAT, PDH, and DLD, thereby disrupting the pyruvate dehydrogenase complex (PDC) and TCA cycle. In vitro and in vivo investigations demonstrated that CuTOT elevates ALP, Runx2, and BMP2 levels, while OMVs induce macrophage polarization toward the M2 phenotype to facilitate osseointegration. These findings suggest that TNZ-OMVs-Cu-TCPP (CuTOT) may serve as an implant surface coating with dual functionality for infection eradication and osteogenesis promotion.¹⁰⁹ MCM represents a copper metal-organic framework (MOF) functionalized with manganese dioxide (MnO₂), which catalyzes the conversion of hydrogen peroxide (H₂O₂) to oxygen (O₂) within biofilms, thereby shifting bacterial metabolism from glycolysis toward aerobic respiration. Western blot analysis demonstrated decreased DLAT protein levels along with reduced expression of marker proteins LIAS and FDX1 in both MCM+NIR and MCM treatment groups, concomitant with observed DLAT oligomerization. Inductively coupled plasma optical emission spectrometry (ICP-OES) quantification of biofilm copper content revealed significantly elevated copper levels in MCM+NIR and MCM groups. RT-qPCR analysis indicated significantly upregulated BMP2, OCN, and RUNX2 gene expression in the MCM group. Comprehensive in vitro studies, infectious arthritis (IAI) models, and bacterial infectious bone defect models confirmed that MCM combined with photothermal therapy exerts antibacterial, osteogenic, and pro-angiogenic effects. This investigation underscores the efficacy of oxygen supplementation in promoting copper-dependent cell death.¹¹⁰ The CpBT nanoreactor was fabricated through the integration of piezoelectric barium titanate (BaTiO₃) with polydopamine (PDA) and copper (Cu). This design leverages PDA-mediated efficient transfer of ultrasonically activated piezoelectric thermal energy to copper, facilitating redox cycling between Cu²⁺ and Cu⁺. Through Cu⁺-catalyzed chemodynamic reactions, endogenous H₂O₂ is converted to hydroxyl radicals (·OH). ROS-induced alterations in membrane permeability significantly enhance intracellular copper influx. The PH-CpBT implant scaffold was constructed by incorporating CpBT nanoreactors, hydroxyapatite (HA), and 3D-printed polyetherketoneketone (PEKK) bone implants. Experimental results demonstrated that PH-CpBT scaffolds disrupt Staphylococcus aureus membrane homeostasis through elevated ROS levels, promoting increased Cu⁺ influx and inducing cuproptosis-like death, thereby exhibiting potent antibacterial efficacy. Transcriptomic and metabolomic analyses revealed that intracellular copper accumulation inhibits the TCA cycle through Cu⁺ binding to lipoylated enzymes (DLAT, GCSH, DBT, DLST), ultimately culminating in cuproptosis-like bacterial cell death. Following implantation of S. aureus-contaminated scaffolds in a rat femoral condyle bone defect model, PH-CpBT scaffolds demonstrated remarkable antibacterial performance and promoted angiogenesis through the release of copper, calcium, and phosphorus ions, thereby enhancing osteogenic differentiation of pre-osteoblasts.¹¹¹ Comprehensive elucidation of cuproptosis mechanisms, combined with the integration of Cu²⁺ into orthopedic implants for cuproptosis modulation to exert antimicrobial effects, represents a promising research direction for reducing postoperative orthopedic infections.

Infectious Wounds and Diabetic Wounds

The carboxyl groups of the Cu-gallic acid-vancomycin (CuGA-VAN) nanozyme complex bind to free amino groups of teichoic acid (TA) in the cell walls of Gram-positive bacteria, enabling rapid capture and targeting of MRSA within the wound fluid microenvironment. Subsequently, the complex exerts oxidase-like (OXD-like) and glutathione peroxidase-like (GSH-Px-like) activities to efficiently generate reactive oxygen species (ROS) for bactericidal action. Concurrently, the nanozyme complex undergoes gradual dissolution in the acidic wound fluid environment, resulting in enrichment of Cu²⁺ and vancomycin (VAN). VAN specifically inhibits bacterial cell wall peptidoglycan synthesis, precipitating cytoskeletal disruption. Cu²⁺ traverses the cell wall and cytoplasmic membrane to enter bacterial cells. Transcriptomic sequencing revealed that Cu²⁺ internalization produces toxic bactericidal effects through disruption of the MRSA respiratory chain, downregulation of genes associated with the TCA cycle and glyoxylate cycle, and induction of lipid peroxide (LPO) accumulation. In vitro experiments demonstrated that the CuGA-VAN nanozyme complex effectively eradicates MRSA infection, promotes proliferation of wound repair cells and angiogenesis, and accelerates wound healing.¹¹² Copper cobalt oxide (CuCo₂O₄) nanoflowers possess multiple enzyme-like activities, including peroxidase-like, oxidase-like, and glutathione peroxidase-like activities, enabling antimicrobial action through ROS generation. In vitro experiments demonstrated that CuCo₂O₄ disrupts biofilms formed by bacteria. Transcriptomic sequencing analysis indicated that intracellular Cu²⁺ accumulation significantly downregulates TCA cycle-associated genes. qPCR results revealed significant downregulation of genes associated with citrate synthase (CS), succinate dehydrogenase complex subunits (SDHA, SDHB, SDHC), and α-ketoglutarate dehydrogenase complex (DLST), suggesting that CuCo₂O₄ suppresses the TCA cycle. Furthermore, cuproptosis induces lipid peroxide (LPO) accumulation. Collectively, CuCo₂O₄ exerts its effects through disruption of TCA cycle and respiratory chain function and induction of bacterial LPO accumulation, with mechanisms analogous to cuproptosis in eukaryotic cells, indicating that CuCo₂O₄ can induce cuproptosis-like processes in bacteria. During infectious burn wound healing, CuCo₂O₄ promotes granulation tissue regeneration, collagen deposition, angiogenesis, and cellular proliferation.¹¹³ Machine learning-based approaches identified cuproptosis signature genes (SLC31A1, ADNP, and DLAT) in diabetic foot patients. Comparative qRT-PCR analysis of gene expression in diabetic foot ulcer (DFU) tissues versus normal tissues and acute wounds revealed that SLC31A1 was significantly downregulated, ADNP was significantly upregulated, and DLAT showed no detectable expression in DFU tissues.⁷⁸ Notably, the functional roles of these genes in DFU pathogenesis remain to be validated through in vitro and in vivo experimental models. The inherent antimicrobial activity of Cu²⁺ has gained widespread recognition; Cu²⁺ can disrupt cellular structures, leading to cell death, while ROS generated through Cu²⁺ induction enhances bacteriostatic activity. As one of the most abundant transition metal ions in the human body, Cu²⁺ plays crucial roles in critical wound healing processes, including collagen synthesis, angiogenesis, and vascular endothelial growth factor induction. Cuproptosis-like cell death represents a novel mechanism underlying Cu²⁺-mediated antimicrobial activity.

Cuproptosis and Inflammation: Cause or Consequence?

The relationship between copper metabolism dysregulation and inflammation in orthopedic diseases involves complex bidirectional interactions that warrant careful consideration. On one hand, inflammatory conditions characteristic of rheumatoid arthritis, osteomyelitis, and implant-associated infections can actively disrupt copper homeostasis. Pro-inflammatory cytokines such as IL-1β and TNF-α have been shown to upregulate CTR1 expression and alter ATP7A/B trafficking, promoting copper accumulation in synovial tissues and infected sites.^69,78 The hypoxic microenvironment in inflamed joints further enhances copper uptake through HIF-1α-mediated transcriptional regulation of copper transporters.⁷⁷ In this context, elevated copper levels and subsequent cuproptosis may represent secondary phenomena driven primarily by inflammatory cascades.

Conversely, compelling evidence supports copper dysregulation as an initiating pathogenic factor. Mendelian randomization studies have established that genetically predicted elevated serum copper levels precede and causally contribute to osteoarthritis risk, independent of inflammatory status.⁹⁰ In intervertebral disc degeneration, oxidative stress-induced SP1 activation upregulates CTR1 transcription, leading to copper overload and cuproptosis before significant inflammatory infiltration occurs.⁷⁸ Furthermore, the specific aggregation of lipoylated TCA cycle proteins by copper ions represents a direct biochemical insult that compromises cellular energy metabolism, potentially triggering inflammatory responses as a downstream consequence.⁹

We propose that the copper-cuproptosis-inflammation axis operates as a self-perpetuating cycle in orthopedic pathologies: initial copper imbalance (whether genetic, metabolic, or iatrogenic from biomaterials) induces mitochondrial dysfunction and cuproptosis, releasing damage-associated molecular patterns (DAMPs) that activate innate immunity; the resulting inflammatory microenvironment further perturbs copper homeostasis, exacerbating cuproptosis and tissue destruction. This conceptual framework suggests that interrupting this cycle at the copper regulation level—through chelators, ionophores, or targeted delivery systems—may offer therapeutic benefits regardless of whether copper alteration was initially causative or consequential.

Conclusions and Perspectives

Cuproptosis represents a recently identified form of regulated cell death. While the molecular mechanism is well-defined in cell culture models, direct in vivo demonstration of cuproptosis in orthopedic disease remains limited. Current evidence relies primarily on: (i) bioinformatic prediction from human tissue transcriptomes; (ii) pharmacological induction (elesclomol + Cu²⁺) in cell lines; and (iii) biomaterial-based copper delivery in rodent infection or tumor models.^{20,53–55,111,112} Notably, genetic mouse models with conditional knockout of FDX1, LIAS, or other cuproptosis regulators in bone-specific cell types are lacking. Similarly, cuproptosis inhibitors (eg, TTM, D-penicillamine) have not been tested in established orthopedic disease models to assess therapeutic efficacy. We therefore characterize the current evidence base as mechanistically robust but clinically preliminary, warranting cautious interpretation and prioritized investment in in vivo validation studies. Although copper ions are known to target lipoylated proteins within the tricarboxylic acid (TCA) cycle, the precise molecular mechanisms remain incompletely elucidated. Further investigation is warranted to determine whether additional metabolic pathways participate in cuproptosis initiation and to clarify how aggregated lipoylated mitochondrial enzymes activate copper-dependent signaling cascades culminating in cell death. Future research should concentrate on the functional roles of cuproptosis-associated cellular signaling pathways and the development of targeted therapeutic strategies. The translation of cuproptosis-targeted interventions from laboratory investigations to clinical applications confronts multiple challenges. Current literature has reported approaches combining targeted drug delivery systems with gene editing technologies to modulate cuproptosis for cancer therapeutics.¹¹⁴ Through targeted drug delivery systems, cuproptosis modulators can be precisely delivered to pathological tissue cells, while gene editing technologies enable the regulation of cuproptosis-related gene expression, synergistically enhancing or attenuating cuproptosis in target cells. In summary, the integration of gene editing technologies with targeted drug delivery systems enables controllable modulation of cellular cuproptosis, thereby achieving anticipated therapeutic efficacy while minimizing impact on normal cells. Given that lesion sites exhibit excessive exosome secretion,¹¹⁵ whether exosome-based strategies can be exploited to target cuproptosis at pathological sites merits further investigation. Copper ions assume a dual role in vivo, serving as essential elements for physiological processes while potentially inducing pathological damage. Therapeutic modalities must be developed that can eliminate pathological copper accumulation without disrupting physiological copper homeostasis, as well as strategies that specifically induce cuproptosis in pathological tissue cells without compromising normal cell viability. Overexpression of specific receptors on cell membranes has prompted the utilization of ligand-modified nanotherapeutic strategies for targeted treatment.¹¹⁵ Advanced drug delivery systems are essential for ensuring precise delivery of copper chelators and copper ionophores to target tissues, facilitating optimal therapeutic outcomes while minimizing adverse effects. While modulating copper homeostasis offers promising therapeutic avenues, several risks require careful evaluation. Copper is essential for normal bone physiology: it serves as a cofactor for lysyl oxidase (collagen cross-linking and bone matrix integrity), ceruloplasmin (iron mobilization for erythropoiesis and oxygen delivery), and SOD1 (antioxidant defense).^40,46,52 Systemic copper chelation could impair these functions, potentially causing bone fragility, anemia, and increased oxidative stress. Conversely, excessive copper supplementation or ionophore delivery may trigger unintended cuproptosis in non-target tissues, particularly the liver (primary copper storage organ), heart (high mitochondrial density), and neurons (high energy demand). The narrow therapeutic window identified in Cuproptosis and Its Interplay with Other Cell Death Modalities further complicates clinical translation. We recommend that future therapeutic strategies prioritize: (i) localized, controlled-release delivery to minimize systemic exposure; (ii) disease-specific biomarkers to identify patients with confirmed copper dysregulation; and (iii) combination approaches using sub-lethal copper doses with synergistic modalities (eg, oxidative stress induction, immune checkpoint blockade) to enhance efficacy while reducing toxicity.^20,107 The clinical application of cuproptosis is constrained by the lack of specific biomarkers. However, several promising candidates merit immediate investigation for translational potential: (i) circulating lipoylated DLAT oligomers, which represent the direct executioner of cuproptosis and have been detected in aggregated form in osteosarcoma and degenerative disc tissues;^78,106 (ii) plasma FDX1 levels or activity, as this mitochondrial reductase is essential for cuproptosis initiation and shows disease-correlated expression changes;^69,78 (iii) the copper-to-ceruloplasmin ratio, which may reflect labile copper availability more accurately than total serum copper;³⁰ and (iv) urinary or synovial fluid levels of Fe-S cluster proteins (eg, NDUFS1, SDHB) as surrogate markers of cuproptosis-induced mitochondrial dysfunction.⁹ Additionally, imaging-based biomarkers using copper-isotope PET tracers (⁶⁴Cu-ATSM) could non-invasively visualize copper accumulation in orthopedic lesions, providing spatial information complementary to blood-based markers. Development of standardized assays for these candidates should be prioritized to enable patient stratification and treatment monitoring. The clinical field urgently requires the development of biomarkers for early detection and disease monitoring. The antimicrobial effects and cytotoxicity of copper ions are dose-dependent, necessitating thorough investigation into concentration control to trigger cuproptosis-like bacterial cell death without damaging normal cells. Objective assessment suggests that cuproptosis-targeted therapies for orthopedic diseases remain in the early preclinical stage, with clinical translation likely 5–10 years away barring breakthrough advances. Several critical gaps must be addressed: (i) Species-specific pharmacodynamics: murine models may not recapitulate human copper metabolism due to differences in ATP7A/B expression patterns and ceruloplasmin regulation; (ii) Biomaterial integration: while copper-doped scaffolds show promise in rodent infection models,^20,54 long-term biocompatibility, copper leaching kinetics, and optimal dosing regimens require large-animal (ovine or porcine) bone defect studies; (iii) Selectivity challenges: no clinically approved copper ionophores or chelators specifically target bone tissue, necessitating development of bone-homing delivery systems (eg, bisphosphonate-conjugated chelators, hydroxyapatite-targeted nanoparticles); and (iv) Safety monitoring protocols: given copper’s essential physiological roles, clinical trials must establish rigorous monitoring for anemia, neuropathy, and bone marrow suppression. Addressing these gaps through multi-disciplinary collaboration between materials scientists, orthopedists, and pharmacologists represents the most efficient path toward first-in-human studies. The concept of cuproptosis offers novel perspectives for the diagnosis and treatment of orthopedic diseases. Extensive research has demonstrated associations between cuproptosis and various orthopedic conditions, including osteosarcoma, rheumatoid arthritis, and osteoarthritis; however, the specific mechanisms through which copper-induced cell death promotes disease progression in these contexts remain obscure. We must explicitly acknowledge the evidentiary limitations pervading this field: the majority of cited studies rely on bioinformatic correlation (transcriptomic profiling, machine learning signatures) or in vitro pharmacological induction using supraphysiological copper concentrations or synthetic ionophores (eg, elesclomol). These approaches, while mechanistically informative, carry limited predictive validity for complex in vivo microenvironments where hypoxia, pH gradients, immune cell infiltration, and mechanical loading interact. Robust validation requires: (i) genetic animal models with cell-specific deletion of FDX1, LIAS, or ATP7A/B in osteoblasts, osteoclasts, or chondrocytes to definitively establish cuproptosis necessity in disease pathogenesis; (ii) prospective clinical cohorts measuring candidate biomarkers (FDX1, DLAT oligomers, labile copper) in synovial fluid, serum, and bone biopsies from patients with OA, RA, and OS, correlated with radiographic progression and histological severity; and (iii) randomized preclinical trials comparing cuproptosis-modulating interventions (copper chelation vs ionophore delivery) against standard-of-care in clinically relevant large-animal models. Without such rigorous validation, cuproptosis risks remaining an intriguing cell culture phenomenon rather than a clinically actionable therapeutic target. The concept of cuproptosis has introduced a novel approach to the diagnosis and treatment of orthopedic diseases. While the foundational molecular mechanisms are compelling, we emphasize that current clinical applicability remains speculative. Numerous studies indicate a connection between cuproptosis and various orthopedic conditions; however, the precise mechanisms through which copper-induced cell death contributes to disease progression are still not fully understood, and the therapeutic potential of targeting this pathway awaits robust in vivo and clinical validation. Future research should focus on optimally regulating copper levels within the context of bone metabolism, developing therapeutic strategies for orthopedic diseases that target cuproptosis, and bridging the translational gap through biomarker development and rigorous preclinical safety assessment.

Funding Statement

This work was supported by Sanming Project of medicine in Shenzhen (SZSM202106019); 2024 High-quality Development Research Project of Shenzhen Bao’an Public Hospital (BAGZL2024024).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest that could have appeared to influence the work reported in this paper.