SINFH accounts for approximately 25 to 50 percent of all osteonecrosis of the femoral head cases, making it one of the most significant complications of long-term glucocorticoid therapy. The condition arises when abnormal bone metabolism, lipid metabolism imbalance, and blood microcirculation disorders in the femoral head lead to trabecular fracture, bone tissue necrosis and collapse, and ultimately hip dysfunction. Despite decades of research, the exact pathogenesis has remained unclear, with bone metabolism disorders, vascular endothelial injury, cell apoptosis, inflammation, and lipid metabolism disturbances considered the main pathological manifestations.

The review’s central conceptual contribution is the distinction between physiological and pathological roles of ROS, which the authors frame as an “oxidative window”—the optimal range of ROS required for normal cellular processes. At low to moderate concentrations, ROS function as essential second messengers generated primarily by NADPH oxidases (NOX enzymes) in response to growth factors and cytokines, regulating cell proliferation, survival, differentiation, migration, and metabolism. For example, BMP-2 activates NOX to induce physiological ROS production that serves as a signaling trigger for osteoblast differentiation, while ROS-mediated activation of ERK1/2 and p38 MAPK pathways is crucial for osteoblast proliferation. In contrast, supraphysiological ROS levels overwhelm cellular antioxidant defenses, causing mitochondrial DNA mutations, lipid peroxidation, protein carbonylation, and irreversible cell damage that triggers bone metabolism disorders, vascular endothelial injury, and apoptosis.

The transition from physiological signaling to pathological oxidative stress depends on the magnitude, duration, and subcellular location of ROS production. This concentration-dependent duality has profound therapeutic implications: the goal in SINFH should not be complete elimination of ROS, but rather restoration of redox homeostasis within the oxidative window. The authors support this with evidence that the antioxidant N-acetylcysteine inhibits mineralization of osteoblast-like MC3T3-E1 cells in a dose-dependent manner, suggesting that physiological levels of ROS are actually required for osteoblast mineralization.

On the bone formation side, the review highlights the complex and sometimes opposing roles of sirtuin family members in regulating oxidative stress and osteogenic differentiation. SIRT6 and SIRT3 function as protective factors: SIRT6 knockout mice showed decreased alkaline phosphatase expression and reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), while promoting SIRT6 expression reduced ROS accumulation and enhanced osteogenic activity. Resveratrol activates the AMPK/PGC-1α/SIRT3 pathway, inhibiting oxidative stress in BMSCs and enhancing Runx2 and osteocalcin expression. In contrast, SIRT2 exerts opposing effects—it induces oxidative stress and promotes BMSC apoptosis, and inhibiting SIRT2 expression alleviates both, slowing SINFH progression. This functional divergence suggests that therapeutic strategies should selectively target specific sirtuin isoforms rather than broadly modulating sirtuin activity.

On the bone resorption side, the review details how oxidative stress drives hyperactive osteoclasts. In necrotic areas of the human femoral head, antioxidant enzymes including catalase, γ-glutamylcysteine synthetase, and superoxide dismutase 1 are significantly downregulated, while osteoclast-related proteins such as cathepsin K and tartrate-resistant acid phosphatase are significantly upregulated. Under oxidative stress conditions, ROS leads to overactivation of RANKL and its receptor RANK, triggering the MAPKs signaling cascade and activating NFATC1, which increases osteoclast differentiation and enhances bone resorption. The Nrf2 transcription factor emerges as a key regulator that can inhibit excessive bone resorption caused by oxidative stress by modulating RANKL-RANK binding, with compounds like Tanshinone I from Danshen shown to increase antioxidant enzyme activity through Nrf2 activation.

The review also examines how oxidative stress accelerates apoptosis of bone cells through the Bcl-2/BAX pathway, with glucocorticoid-induced ROS increasing endoplasmic reticulum stress and mitochondrial DNA damage. Markers of DNA oxidative damage such as 8-hydroxy-2′-deoxyguanosine (8-OHdG) are positively correlated with bone cell apoptosis in SINFH patients, and antioxidants like diphenyleneiodonium and N-acetylcysteine can reduce 8-OHdG levels and reverse dexamethasone-mediated bone cell apoptosis. Vitamin E has been shown to reduce caspase-3 expression while upregulating Bcl-2, alleviating DNA oxidative damage in bone marrow hematopoietic cells.

Vascular injury represents another critical axis. Oxidative stress causes structural and functional impairments of vascular endothelial cells, increases the risk of thrombosis, and exacerbates ischemia and hypoxia in the femoral head. Li and colleagues found that two weeks after steroid injection, the perfusion frequency of small vessels in the femoral head of rats was significantly reduced, with extensive oxidative damage in bone marrow vessels; the antioxidant edaravone significantly reduced this damage and increased small vessel perfusion. Thymoquinone has been shown to reduce malondialdehyde levels and increase glutathione levels, alleviating oxidative stress-induced vascular endothelial injury, while hydrogen-rich saline increased GSH levels and microvascular density in an experimental rabbit model.

Perhaps the most novel aspect of the review is its treatment of the bidirectional relationship between lipid metabolism disturbances and oxidative stress. Glucocorticoids profoundly affect lipid metabolism by promoting adipogenic differentiation of BMSCs at the expense of osteogenic differentiation, resulting in intraosseous lipid accumulation, adipocyte hypertrophy, and increased marrow fat content that mechanically compresses sinusoids and impairs microcirculation. The accumulated lipids serve as substrates for lipid peroxidation, generating malondialdehyde and 4-hydroxynonenal—products that are not merely passive markers but active mediators that form covalent adducts with proteins, induce mitochondrial dysfunction promoting further ROS production, and activate inflammatory signaling pathways. Conversely, lipid accumulation in bone marrow adipocytes and osteoblasts induces endoplasmic reticulum stress and mitochondrial dysfunction, both potent sources of ROS, while oxidized low-density lipoproteins accumulated in bone tissue can activate NOX enzymes, amplifying ROS production. This creates a vicious cycle: glucocorticoid-induced lipid accumulation triggers oxidative stress, which promotes lipid peroxidation, which exacerbates oxidative damage and inflammation, leading to further tissue injury.

The review also maps the crosstalk among oxidative stress-mediated pathological processes, identifying self-amplifying loops that connect vascular injury, inflammation, apoptosis, and lipid metabolism. Vascular injury induced by oxidative stress leads to ischemia and hypoxia, which in turn exacerbates oxidative stress through mitochondrial electron transport chain dysfunction during reoxygenation, hypoxia-inducible factor-mediated NOX expression, and depletion of cellular antioxidant reserves. ROS activates NF-κB, a master transcription factor for pro-inflammatory cytokines including TNF-α, IL-1β, and IL-17, which not only promote osteoclast activation but also stimulate further ROS production from macrophages and other immune cells. Apoptotic cells release damage-associated molecular patterns that activate inflammatory responses and promote osteoclast differentiation, creating a situation where bone resorption exceeds formation and accelerating structural deterioration.

On the signaling pathway front, the review comparatively evaluates the Nrf2/Keap1, Wnt/β-catenin, Akt, and JNK/c-Jun pathways for their relative importance and therapeutic potential. The Nrf2/Keap1 pathway emerges as most promising for intervention due to its central role as the master regulator of the antioxidant response, the availability of pharmacological activators with established safety profiles, and its integration with other pathways. The Akt pathway ranks second given its unique position simultaneously regulating osteogenesis via Runx2, angiogenesis via HIF-1α/VEGF, apoptosis via Bad/Bcl-2, and oxidative stress via Nrf2/HO-1. Astragaloside IV, an extract from Astragalus, has been shown to inhibit cell apoptosis and oxidative stress through the Akt/Bad/Bcl-2 and Akt/Nrf2/HO-1 pathways, preventing glucocorticoid-induced SINFH. The JNK/c-Jun pathway, though clearly involved in oxidative stress-induced apoptosis, presents challenges for therapeutic targeting due to its context-dependent effects.

Despite promising preclinical results, the clinical translation of antioxidant therapy for SINFH faces multiple bottlenecks. Many potent antioxidants such as resveratrol and curcumin exhibit poor oral bioavailability and undergo rapid metabolism, limiting their therapeutic efficacy. Achieving therapeutic concentrations specifically within the femoral head remains challenging due to the site’s limited vascular access, though local delivery systems including hydrogels, 3D-printed scaffolds, and nanoparticles offer potential solutions requiring optimization. The optimal timing of antioxidant intervention—whether before, during, or after glucocorticoid administration—remains undefined, as pre-treatment might prevent initial oxidative damage but could interfere with the glucocorticoid’s therapeutic effects, while post-treatment might arrive too late after irreversible damage has occurred. Current clinical markers of oxidative stress, including malondialdehyde, superoxide dismutase, and glutathione, suffer from poor specificity influenced by diet and circadian rhythm, lack of tissue specificity, and diurnal variation, though emerging biomarkers such as oxidized LDL, F2-isoprostanes, and protein carbonyls offer improved specificity pending validation in SINFH cohorts.

Given the multifactorial nature of SINFH pathogenesis, the authors argue that antioxidant monotherapy may be insufficient, and rational combination strategies targeting multiple pathological nodes simultaneously warrant investigation. These include combining antioxidants with angiogenic factors to address both oxidative damage and vascular insufficiency, with anti-inflammatory agents to break the inflammation-ROS cycle, with bone anabolic drugs to promote bone formation while protecting against oxidative damage, and with lipid-lowering agents possessing antioxidant properties to target the lipid metabolism-oxidative stress axis. The review ultimately concludes that maintaining a dynamic balance between oxidation and antioxidation may be more important than completely suppressing redox reactions, positioning oxidative stress regulation as a promising new direction for the prevention and treatment of this increasingly recognized complication of steroid therapy.

Subject of Research: Oxidative stress mechanisms in steroid-induced necrosis of the femoral head

Article Title: Oxidative stress in steroid-induced necrosis of femoral head: a review revealing new insights into pathogenic mechanisms

Article References: Wu, Y., Ma, R., & Jiang, H. (2026). Oxidative stress in steroid-induced necrosis of femoral head: a review revealing new insights into pathogenic mechanisms. Frontiers in Musculoskeletal Disorders, 4, Article 1788598. https://doi.org/10.3389/fmscd.2026.1788598

Image Credits: AI Generated

DOI: 10.3389/fmscd.2026.1788598

Keywords: oxidative stress, steroid-induced necrosis of femoral head, reactive oxygen species, antioxidants, Nrf2 signaling, osteonecrosis, glucocorticoids, bone metabolism, apoptosis, vascular injury, lipid metabolism, inflammation

The research team, working at the Korea Research Institute of Bioscience and Bioscience-affiliated laboratories, took an approach that sidesteps a long-standing obstacle in the field. Bone marrow from children with Legg-Calvé-Perthes disease is scarce and difficult to study, and even when mesenchymal stromal cells, the body’s principal skeletal progenitors, are isolated from osteonecrotic bone, they carry the imprint of a diseased, inflamed, poorly perfused tissue environment. Any abnormality observed in such cells could reflect that hostile microenvironment rather than an intrinsic flaw. To separate the two, the researchers reprogrammed skin fibroblasts from two patients with the disease, and from a healthy donor, into induced pluripotent stem cells using a non-integrating Sendai virus system that delivers the reprogramming factors OCT4, SOX2, KLF4 and c-MYC. Because skin cells can be reset to an embryonic-like state and then steered into mesenchymal stem cells under identical, standardized conditions, any difference that emerges between patient-derived and healthy cells must be written into the cells themselves rather than imposed by the tissue around them.

The quality control on this cellular reset was rigorous. The resulting induced pluripotent stem cell lines formed compact colonies with the characteristic morphology of embryonic stem cells, stained strongly for alkaline phosphatase, and expressed the canonical pluripotency markers OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 at levels comparable across all three lines. Quantitative PCR confirmed expression of OCT4 and REX1 in the undifferentiated state, and when the cells were allowed to spontaneously differentiate into embryoid bodies, they activated ectodermal markers such as SOX1 and PAX6, mesodermal markers including HAND1 and PDGFRA, and endodermal markers SOX7 and AFP. Immunostaining for TUJ1, NESTIN, DESMIN, alpha-SMA, FOXA2 and SOX17 confirmed protein-level differentiation into all three germ layers, and teratoma assays in immunodeficient mice produced tissues representing ectoderm, mesoderm and endoderm. G-band karyotyping showed normal diploid chromosomes in every line, ensuring that any phenotype found later was not an artefact of culture stress or genomic instability.

With validated stem cell lines in hand, the team differentiated them into induced mesenchymal stem cells using a defined, xeno-free protocol, first patterning the cells toward the mesodermal lineage for four days and then maturing them for a further seventeen days on fibronectin-coated plates. Flow cytometry confirmed that the resulting cells expressed the canonical mesenchymal markers CD73 and CD105 and lacked the haematopoietic markers CD34 and CD45, consistent with bona fide MSC identity. One subtlety stood out: the two patient-derived lines expressed CD90, a surface glycoprotein involved in adhesion and MSC immunophenotype, at only about sixty to seventy percent of the level seen in healthy donor cells, which reached roughly ninety-five percent. Proliferation rates, however, were indistinguishable between the groups, meaning the patient cells grew normally even as they showed this subtle phenotypic deviation, hinting that the real differences lay deeper, in what the cells could become rather than how fast they could multiply.

What the cells could become turned out to be the central finding. Under osteogenic induction, healthy donor cells produced abundant mineralized matrix, revealed by intense Alizarin Red S staining, and strongly upregulated the osteoblast genes RUNX2 and BGLAP. Both patient-derived lines formed dramatically fewer mineral deposits and mounted only weak induction of the same genes. Adipogenesis fared no better: healthy cells accumulated plentiful lipid droplets stained by Oil Red O and induced the adipogenic regulators PPARG and ADIPOQ, while patient cells showed markedly reduced lipid accumulation and gene induction. Chondrogenesis, assessed by Alcian Blue staining of sulfated glycosaminoglycans and by expression of SOX9 and COL2A1, was also reduced but comparatively better preserved. Crucially, this pattern of a simultaneous, broad suppression of all three mesenchymal lineages argues against the classic idea of a simple lineage shift, in which bone-forming cells are diverted into fat cells. Instead, it points to a deeper loss of overall differentiation competence within the patient-derived progenitors themselves.

The molecular culprit emerged when the researchers examined the Wnt/beta-catenin pathway, the master regulator of mesenchymal lineage commitment. Beta-catenin, the transcriptional co-activator at the heart of canonical Wnt signalling, promotes osteogenesis and chondrogenesis while restraining adipogenesis, and its intracellular abundance is normally kept in check by glycogen synthase kinase-3, which phosphorylates beta-catenin and tags it for proteasomal destruction. In the patient-derived cells, the team found a striking double abnormality. Transcripts for CTNNB1, the gene encoding beta-catenin, were significantly reduced, while GSK3B mRNA was markedly elevated. At the protein level, phosphorylation of GSK3alpha at Tyr279 and GSK3beta at Tyr216, the modifications associated with enhanced enzymatic activity, was substantially increased, reaching roughly 1.8- to 2.2-fold above healthy donor levels in the more severely affected patient line. Total beta-catenin protein and intracellular beta-catenin fluorescence were correspondingly diminished, painting a coherent picture of a kinase pushed into overdrive and a key transcriptional regulator destroyed faster than it should be.

The disease signature extended into inflammation, a domain long suspected to matter in this condition because children with active disease show elevated IL-6 and HMGB1 in synovial fluid. Even without any inflammatory stimulus, the patient-derived mesenchymal cells expressed higher baseline levels of IL6, PTGS2 and CCL2, indicating a primed inflammatory state. When the cells were challenged with IL-1beta, the patient cells responded with larger fold increases in these cytokines than healthy cells, and the Wnt target genes AXIN2 and LEF1, already slightly reduced at baseline, were driven down even further. Western blotting showed that IL-1beta pushed GSK3 phosphorylation higher in patient cells than in controls while beta-catenin, already low, declined still more. These exaggerated responses persisted after osteogenic differentiation, where patient-derived osteoblasts displayed stronger NF-kappaB phosphorylation and greater degradation of its inhibitor I-kappa-B-alpha upon inflammatory stimulation. Transcriptomic sequencing reinforced the theme, revealing enriched alterations in inflammatory signalling, extracellular matrix organization and Wnt-related regulatory pathways in both patient fibroblasts and patient-derived mesenchymal cells, with upregulation of multiple extracellular Wnt antagonists including DKK1, SFRP1, SFRP2, WIF1 and SOST.

The most striking result, and the one with the clearest translational implication, came next. When the researchers treated the patient-derived cells with lithium chloride, a long-established inhibitor of GSK3, beta-catenin fluorescence recovered to levels comparable to untreated healthy donor cells, with the rescue being strongest precisely where the deficit had been greatest. More importantly, the functional defect yielded to the same treatment. Lithium chloride substantially increased mineralized nodule formation and Oil Red O staining in both patient lines during osteogenic and adipogenic induction, and quantitative PCR confirmed that either lithium chloride or the more selective inhibitor CHIR99021 significantly boosted expression of BGLAP, RUNX2, ADIPOQ and PPARG. The differentiation programs had not been deleted; they had merely been silenced by an overactive kinase, and silencing the kinase switched them back on. This established that the mesenchymal failure in Legg-Calvé-Perthes disease reflects a reversible, kinase-dependent signalling imbalance rather than irreversible cellular damage.

The study’s authors are careful to place their findings in context. Observations in patients, such as elevated circulating leptin and increased adipose tissue within the affected femoral head, reflect systemic and tissue-level changes that arise from ischemia, inflammation and altered marrow mechanics, and the in vitro reduction in adipogenesis seen here does not contradict that clinical picture. Rather, the platform isolates the intrinsic cellular component of the disease, complementing animal models in which ischemia was surgically induced in piglets and rodents. Those models have shown that ischemia activates hypoxia-inducible factor 1-alpha and drives inflammatory and angiogenic responses, and the new human data now connect those tissue-level events to a specific, druggable intracellular node inside the progenitors that must rebuild the bone. The authors propose a reciprocal feedback loop in which inflammatory activation sustains GSK3 activity, further destabilizing beta-catenin and progressively compromising skeletal regeneration.

For a disease whose current treatments range from conservative containment to major reconstructive surgery, the prospect of a pharmacological strategy that restores the bone-building capacity of a child’s own progenitor cells is genuinely exciting. Lithium is an old drug with a well-characterized safety profile in other contexts, and the iPSC-based model developed here provides a patient-specific testing ground for optimizing GSK3-targeted regenerative therapies before they reach the clinic. Much work remains, including validating the findings in larger patient cohorts and determining how a GSK3 inhibitor could be delivered safely to the developing femoral head. But the central message stands: the cellular failure underlying a devastating childhood hip disease is not a life sentence written into the genome, but a reversible regulatory state, and with the right key, the machinery of skeletal repair can be switched back on.

Subject of Research: Reversible GSK3/beta-catenin dysregulation in patient-derived iPSC-mesenchymal stem cells from children with Legg-Calvé-Perthes disease

Article Title: Patient‐Derived iPSC‐MSC Modelling Reveals Reversible GSK3‐Mediated Suppression of Mesenchymal Lineage Differentiation in Legg‐Calvé‐Perthes Disease

Article References: Seol, B., Lim, H. J., Song, C. L., Lee, J., & Cho, Y. S. (2026). Patient‐Derived iPSC ‐ MSC Modelling Reveals Reversible GSK3 ‐Mediated Suppression of Mesenchymal Lineage Differentiation in Legg‐Calvé‐Perthes Disease. Journal of Cellular and Molecular Medicine, 30(17), Article e71350. https://doi.org/10.1111/jcmm.71350

Image Credits: AI Generated

DOI: 10.1111/jcmm.71350

Keywords: Legg-Calvé-Perthes disease, induced pluripotent stem cells, mesenchymal stem cells, GSK3, beta-catenin, Wnt signalling, osteonecrosis, osteogenesis, inflammation, lithium chloride, CHIR99021, paediatric orthopaedics