Bone is a dynamic and metabolically active tissue that maintains a delicate balance between anabolic and catabolic processes throughout an individual’s life to preserve its structural integrity and strength. Bone remodeling is an ongoing process that occurs in response to mechanical stress, primarily involving bone resorption, facilitated by osteoclasts, and bone formation, mediated by osteoblasts. The dysfunction of such processes significantly contributes to various bone tissue diseases (Siddiqui and Partridge, 2016). BMSCs possess the remarkable ability for self-renewal and differentiation into various cell types. Notably, they can differentiate into osteoblasts, adipocytes, and chondrocytes (Pierce et al., 2019). Osteogenic/adipogenic differentiation disorder of BMSCs is the primary underlying cause of ONFH (Chen et al., 2020).

Recent studies have highlighted the impact of DNA methylation on osteogenic differentiation and its involvement in regulating bone metabolism (Malvicini et al., 2019; Li et al., 2019). Sun et al investigated the role of DNA methylation in steroid-induced osteonecrosis of femoral head (SONFH). Their results demonstrated that the proliferation ability of BMSCs in patients with SONFH was weakened. Additionally, the transcription level of the ABCB1 gene in BMSCs decreased, whereas its methylation modification level increased. The P-glycoprotein, encoded by this gene, played a crucial role in drug absorption and distribution. Notably, oral glucocorticoids served as the protein’s substrates. When BMSCs were treated with 5-aza, the expression of ABCB1 was rapidly restored (Sun et al., 2013). Further research by the same study group in the same patient population indicated that icariin-treated BMSCs reduced the methylation modification level of the CpG island of ABCB1, increased its gene expression, and promoted the osteogenic differentiation of BMSCs (Sun et al., 2015). Lysine-specific demethylase 5A (KDM5A) negatively regulates BMSC osteogenic differentiation by modulating H3K4me3 levels on the promoters of key osteogenic genes (Runx2, OCN, OPN) in SONFH (Yan et al., 2024). Human umbilical cord MSCs-derived exosomes (hucMSCs-exos) ameliorated alcohol-induced ONFH by inhibiting miR-25-3p DNA methylation and GREM1 expression in BMSCs (Tang et al., 2025). Dexamethasone trigger MC3T3E1 cells ferroptosis by promoting DNMT3a-mediated DNA methylation and downregulating Sirt1 expression in SONFH (Xiao et al., 2025).

DNA methylation has emerged as a potential therapeutic target for osteonecrosis of ONFH (Sun et al., 2015). A study on gene polymorphisms in the Chinese Han population with SONFN validated that polymorphisms in the ABCB1 and CYP450 genes were associated with the risk of steroid-induced ONFN. In particular, the CpG loci within the ABCB1 gene (ABCB1-1-192, ABCB1-2-32, and ABCB1-2-43) were identified with significantly different methylation levels (Huang et al., 2020). As a receptor within the Wnt signaling pathway, frizzled 1 (FZD1) is fundamental in osteoblast differentiation. In BMSCs from SONFH patients, hypermethylation of the FZD1 promoter CpG island reduced the expression of FZD1. Consequently, this inhibition affects the Wnt signaling pathway, weakens the proliferation ability of BMSCs, and hinders osteogenic differentiation, while also promoting adipogenic differentiation. Nonetheless, treatment with 5-aza can restore FZD1 expression by decreasing its methylation level, ultimately facilitating osteogenic differentiation and potentially serving as a therapeutic approach (Wu F. et al., 2019).

The inhibitory effect of the inflammatory factors such as TNF-α and IL-6, which are widely recognized as the main cytokines promoting apoptosis of bone cells or disturbance in bone metabolism associated with ONFH (Cheng et al., 2023; Ozawa et al., 2024), on osteogenic differentiation of BMSCs through epigenetic mechanisms remains a critical concern. The previous studies elucidated that the self-methylation of TNF-a and IL-6 plays a vital role in diseases such as obesity (Boonrong et al., 2024) and cognitive impairment (Rump and Adamzik, 2022). Boonrong et al. demonstrated that the hypomethylation in the TNF-α promoter correlated with elevated TNF-α levels and metabolic dysfunction in obese individuals, suggesting a feedforward loop between inflammation and epigenetic dysregulation. Furthermore, the hypermethylation of IL-6 regulated by DNMT1, contributed to the depression-like behaviors through inducing neuroinflammation in mice (Bai et al., 2023). An early study reported that TNF-α elevated the methylation level of the CpG region of Runx2, leading to the inhibition of the Wnt signaling pathway, subsequently hindering the differentiation of BMSCs into osteoblasts (Fang et al., 2019). However, the role of DNA methylation of TNF-α and IL-6 in ONFH is still unclear.

In conclusion, DNA methylation obstructs the BMSC-osteoblast differentiation by affecting the expression of osteogenic differentiation-related genes. Conversely, DNA demethylation reagents such as 5-aza-dc and icariin, can lower the methylation level of osteogenic genes, supporting osteogenic differentiation, and may potentially be developed as therapies (Figure 2).

Bone, a richly vascularized connective tissue, serves as the main source of oxygen, nutrients, hormones, neurotransmitters, and growth factors for bone cells. The vasculature is indispensable for appropriate bone development, regeneration, and remodeling (Peng et al., 2020). Blood vessels in bone develop through the process of angiogenesis. Their functional impairment can lead to various orthopedic diseases, including osteoporosis and osteonecrosis (Huang B. et al., 2016; Filipowska et al., 2017). In SONFH, angiogenesis may be involved in the repair of femoral head necrosis. Deferoxamine (DFO) can enhance angiogenesis and bone repair by upregulating the expression of key factors, including hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) (Li et al., 2015).

Xu et al. demonstrated that treating BMSCs the DNA methylation inhibitor 5-aza significantly increased the expression of endothelial markers, including CD31/PECAM1, CD105/ENG, and eNOS/VE-cadherin, thereby promoting angiogenesis (Xu R. et al., 2018). In their study using a tibial fracture model, Wang et al. observed that the expression of DNA methyltransferase Dnmt3b peaked on day 10 days after fracture and disappeared by day 28. Knocking out Dnmt3b led to delayed fracture repair, along with reduced numbers and volume blood vessels within the bone. Additionally, in vitro experiments showed that inhibiting Dnmt3b expression in human umbilical-vein endothelial cells resulted in reduced angiogenesis and inhibited endothelial cell migration. The mechanism of action is similar to that of CXCL12 and osteocalcin (osteopontin, OPN). The degree of methylation modification in the promoter region is associated with this process (Wang C. et al., 2018). These aforementioned results suggest that DNA methylation-mediated angiogenesis plays a substantial role in bone repair.

In their study, Yuan et al. conducted microRNA sequencing on the necrotic and normal parts of the femoral head. They found that miR-210 was significantly differentially expressed in necrotic tissue, with increased expression. Notably, when vascular endothelial cells were treated with demethylation reagents, the expression level of miR-210 further increased. Additionally, several angiogenesis-related genes (including VEGF, bFGF, TNF-α, and PCNA) were up-regulated, indicating that miR-210 facilitates angiogenesis and may have a potential therapeutic effect in SONFH (Yuan et al., 2016).

Apoptosis refers to the programmed cell death regulated by genes. As scholars continue to investigate femoral head necrosis, the theory of apoptosis is gradually gaining recognition. Weinstein and their colleagues revealed a significant presence of osteocyte apoptosis in femoral head specimens from 14 SONFH patients (Weinstein et al., 2000). After extensive in vitro and in vivo experiments, the link between steroid- and alcohol-induced apoptosis of bone cells has been firmly established. Osteocyte apoptosis disrupts the sensory function of the cell mechanical network, leading to thinning and sparse trabecular bone. Ultimately, this process contributes to irreversible femoral head collapse. Nonetheless, inhibiting this mechanism can partially mitigate the progression of femoral head necrosis. The transcription factor p53 is vital in this context, activating both death receptor (FAS/TRAILR2)- and mitochondrial (BAX/BAK/BID/NOXA/PUMA/APAF1)-mediated apoptosis pathways by modulating the expression levels of binding site genes (Vousden and Lu, 2002). Abnormal hypermethylation in the promoter region of the p53 gene reduces the gene expression level, thereby affecting cell apoptosis. Moreover, apoptosis is also regulated by the impact of DNA methylation on the expression levels of Fas, Caspase-8, and p14ARF (Gopisetty et al., 2006).

TET3 is responsible for catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmc), substantially contributing to DNA demethylation (Wang et al., 2022; Zhang et al., 2023), which may play a crucial role in biological processes of ONFH by affecting gene expression (Wang et al., 2022). Early in vivo and in vitro studies demonstrated that the expression of TET3-5hmC increased in bone tissue associated with human steroid-induced ONFH and that the elevated TET3-5hmC levels correlated with weakened proliferation and increased apoptosis of osteocytes induced by steroid (Rath et al., 2024). These apoptosis-promoting effects of TET3-5hmC were medicated by PI3K/Akt signaling pathway. The knockdown on TET3 expression could counteract the inhibitory effect of steroid on the Akt pathway and suppress osteocyte apoptosis. On the other side, some studies have suggested that TET3 may promote repairing of spinal cord injury (Zhang et al., 2024) as shown by that TET3-mediated demethylation reshaped the methylation patterns of human umbilical cord mesenchymal stem cells (HUCMSCs), enabling their efficient one-step conversion into oligodendrocyte precursor cells (OPCs) for repairing spinal cord injury. Notably, this functional dichotomy may stem from tissue-specific gene regulatory network. These apoptosis-promoting and repair-promoting effects highlight the complexity in biological roles of TET3, suggesting that its therapeutic targeting might require precise spatiotemporal regulation. The TET3-5hmC-Akt axis represents a promising target of early intervention for steroid-associated ONFH.

In addition to subchondral osteonecrosis and collapse, the pathological process of femoral head necrosis also includes cartilage damage, degeneration and necrosis. In general, articular cartilage damage occurs during the early stages of ONFH and can lead to hip joint instability and femoral head collapse, further contributing to disease progression. Timely treatment of hip cartilage injuries can delay such progression and extend the window for surgical intervention (Xu et al., 2017; Zhao et al., 2017). Although chondrocytes do not undergo a remodeling process, DNA methylation remains vital in regulating chondrogenesis and maintenance, which is achieved by modulating the expression of chondrogenesis-related genes, including GDF5, SOX9, and MMP13 (Ramos and Meulenbelt, 2017). Moreover, during the differentiation of BMSCs into chondrocytes, the methylation level of the CpG site in the COL10A1 gene promoter region decreases. This demethylation process is associated with an increase in COL10A1 expression, ultimately promoting the differentiation of BMSCs into chondrocytes (Zimmermann et al., 2008).

A genome-wide DNA methylation sequencing study of hip articular cartilage in individuals with femoral head necrosis and femoral neck fracture revealed 480 hypermethylated sites and 1,335 hypomethylated sites associated with femoral head necrosis. Among these, the DNA methylation levels of several genes, namely, PDE4D, CARS, RUNX2, ADAMTS12, and LRP5, were decreased. Subsequent immunohistochemical experiments confirmed significant increased protein expressions of ADAMTS12, CARS, PDE4D, LRP5, and RUNX2 in the necrotic cartilage of the femoral head. Methylation yields a critical biological impact by influencing the methylation status of relevant genes in articular cartilage cells, hence regulating gene expression and exerting functional effects. Notably, cysteinyl-tRNA synthetase (CARS) has emerged as the most statistically distinct gene in DNA methylation sequencing results, encoding an aminoacyl-tRNA synthetase and is involved in regulating mitochondrial behaviors. Considering its potential in cartilage injury, further study is required to explore the role of CARS in ONFH-associated mitochondrial dysfunction (Wu J. et al., 2019). A recent study involving multiomics integration revealed 22 dysregulated genes in ONFH cartilage. Critically, MMP13 exhibited promoter hypomethylation coupled with transcriptional upregulation, driving extracellular matrix (ECM) degradation through collagen II cleavage. Conversely, GDF10 and CHI3L1 suppression was linked to promoter hypermethylation, leading to inflammatory changes and impairing BMP-mediated chondrogenesis. Dexamethasone induced DNA methylation changes, dysregulating effector molecules (MMP13, GDF10, and CHI3L1 et al.). This drives ECM degradation and impairs bone repair, culminating in femoral head collapse (Lu et al., 2024).

Genes involved in DNA methylation modifications are crucial in signaling pathways, affecting the abnormal differentiation of BMSCs in ONFH, maturation and differentiation of osteoclasts, and osteocyte apoptosis. The classical Wnt/β-catenin pathway significantly influences bone metabolism, generating dual effects: inhibiting the differentiation of BMSCs into chondrocytes and adipocytes, while promoting their differentiation into osteoblasts (Li et al., 2018; Hayashi and Nakashima, 2019).

The FZD protein, a 7-transmembrane receptor in the Wnt signaling pathway, interacts with Wnt ligands to activate downstream signaling. In particular, FZD1 has been implicated in promoting osteoblast differentiation and mineralization. Wu et al. they investigated DNA methylation in the promoter region of the FZD1 gene using BMSCs from patients with hormonal osteonecrosis of the femoral head. Notably, the FZD1 mRNA and protein levels were significantly reduced, inhibiting the Wnt/β-catenin signaling pathway and suppressing BMSC osteogenic differentiation, while also promoting adipogenic differentiation. However, treatment with the demethylation reagent 5-aza restored FZD1 expression, activating the Wnt/β-catenin pathway and ultimately enhancing BMSC osteogenic differentiation while inhibiting adipogenic differentiation (Wu F. et al., 2019). The osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-Β ligand (RANKL)/RANK pathway remains essential in osteoclast maturation and differentiation. RANKL binds to RANK on the surface of osteoclasts, promoting their differentiation. OPG, secreted by osteoblasts, competes with RANKL, preventing RANKL from binding to RANK and inhibiting osteoclast function. Normally, OPG/RANKL/RANK is well-balanced. However, excessive alcohol and hormone intake can disrupt this balance, leading to osteoclast activation and reduced bone mass. Abnormal hypermethylation sites in the three genes have been observed in patients with hormonal and alcoholic femoral head osteonecrosis. The degree of OPG/RANKL/RANK hypermethylation may serve as a diagnostic marker for alcoholic femoral head osteonecrosis (Sun et al., 2021; Wang et al., 2021). The Akt signaling pathway has a pivotal impact on attenuating osteocyte proliferation through 3-5 hmC modification mediated by TET enzymes, eventually leading to apoptosis. Additionally, KEGG signaling analysis of osteocyte hMeDIP-5 hmC sequencing revealed that apart from Akt, the Notch and Wnt/β-catenin pathways are associated with hormone-induced DNA demethylation. In particular, Notch signaling promotes osteogenic differentiation, blood vessel formation, and bone formation (Ramasamy et al., 2014; Zhao et al., 2017).