Osteonecrosis (ON) is a debilitating skeletal disorder characterized by ischemic bone cell death and structural collapse, arising from multifactorial etiologies, including vascular injury, mechanical stress, and metabolic dysregulation (Konarski et al., 2022; Keating, 2006; Glueck et al., 2008). While traumatic events are well-recognized contributors to ON pathogenesis, nontraumatic cases often idiopathic remain poorly understood, implicating complex interactions between genetic susceptibility, lipid metabolism dysregulation, and coagulation abnormalities (Hines et al., 2021; Chang et al., 2020; Seamon et al., 2012; Sekiya et al., 2010). Notably, dyslipidemia, defined by elevated triglycerides (TG), increased low-density lipoprotein cholesterol (LDL-C), and reduced high-density lipoprotein cholesterol (HDL-C), has been consistently associated with ON progression. Clinical evidence indicates that aberrant lipid profiles are prevalent in idiopathic, steroid-induced, and alcohol-associated ON, suggesting that lipid accumulation may compromise intraosseous blood flow, thereby exacerbating ischemia and necrosis (Santos et al., 2016; Borén et al., 2020; Motta et al., 2022). However, whether lipid abnormalities are causal drivers of ON or merely secondary consequences remains unresolved, underscoring the need for causal inference methodologies to disentangle these complex relationships (Ottensmann et al., 2023; Fahy et al., 2009).

Advances in lipidomics have revolutionized our understanding of lipid diversity, enabling precise quantification of lipid species such as phosphatidylcholines (PCs), triacylglycerols (TAGs), and cholesterol esters (CEs) (Hornburg et al., 2023; Levy et al., 2001). These lipids regulate critical biological processes, including redox homeostasis, inflammatory signaling, and energy metabolism, with dysregulation implicated in cardiometabolic diseases, neurodegeneration, and aging (Serhan, 2014; van Meer et al., 2008; Yap et al., 2023; Eichelmann et al., 2022). Emerging evidence suggests specific lipid subspecies, rather than broad lipid classes, may serve as biomarkers or mediators of ON (Rahman et al., 2023; Wang et al., 2021a). Yet, existing studies predominantly focus on conventional lipid panels (e.g., HDL-C, LDL-C), leaving the role of lipidomic complexity in ON largely unexplored.

Mendelian randomization (MR), a genetic instrumental variable approach, offers a robust framework to infer causality by leveraging genetic variants as proxies for modifiable exposures (Yu et al., 2024; Yu et al., 2023). Unlike observational studies prone to confounding and reverse causation, MR minimizes these biases through the random allocation of alleles during gamete formation (Yan et al., 2022; Kabata et al., 2005). While prior MR studies have linked lipid traits to musculoskeletal disorders (Weinstein et al., 2000; Motta et al., 2022), no study has systematically evaluated the bidirectional causal relationships between detailed lipidomic profiles and ON. Furthermore, the molecular mechanisms underlying lipid-ON interactions, as well as the target genes involved (e.g., oxidative stress, osteoblast dysfunction, or vascular impairment), remain speculative, necessitating integrative multiomics investigations.

In this study, we employ bidirectional two-sample MR to assess causal links between 179 lipid species and ON risk using large scale genome wide association study (GWAS) data. We further integrate transcriptomic profiling of osteonecrotic cartilage to validate candidate genes and pathways. Our findings reveal bidirectional lipid-ON associations, identify protective and pathogenic lipid subspecies, and uncover key regulatory networks such as APOE. This work advances our understanding of lipid-driven ON pathogenesis and provides actionable targets for therapeutic intervention.

ON, a devastating condition marked by ischemic bone death, remains etiologically enigmatic despite associations with vascular disruption, mechanical stress, and metabolic dysfunction (Lespasio et al., 2019; Mankin, 1992; Chang et al., 1993). A major strength of our study lies in its integrative multi-omics framework, which combines bidirectional MR analysis, transcriptomic profiling, and experimental validation. This systems-level approach allows us to triangulate evidence across genetic, transcript, and protein levels, thereby enhancing the robustness and biological relevance of our findings. By leveraging multiple independent data modalities, we provide a comprehensive and multidimensional view of the lipid-osteonecrosis axis that would not be achievable through a single method alone.

The relationship between lipidomes and ON is significant. Elevated levels of blood lipids, such as cholesterol and triglycerides, are linked to the development of ON. Abnormal lipid metabolism can lead to the formation of fat emboli, which obstruct blood vessels and reduce blood flow to the bone, causing ischemia and subsequent bone cell death (Yu et al., 2024; Yu et al., 2023). Studies have demonstrated that lipid abnormalities are prevalent in patients with nontraumatic ON, indicating a direct link between dyslipidemia and the disease (Wang et al., 2021b). Research has shown that managing lipid levels through lifestyle changes and medications can help prevent or slow the progression of ON. For instance, changes in bone tissue lipids are significant in individuals with steroid- and alcohol-induced ON (Brett et al., 2021). Another study emphasized the role of abnormal lipid profiles in the development of nontraumatic ON, highlighting the importance of monitoring and managing blood lipid levels to mitigate the risk (Yan et al., 2022).

We identified phosphatidylcholines (PCs) as protective lipid species, with PC (18:1_18:1), PC (O-16:0_16:1), and PC (O-18:0_16:1) inversely associated with ON risk. Some studies have reported that PCs are critical for membrane integrity, lipid transport, and anti-inflammatory signaling (Kent, 2005; Gibellini and Smith, 2010; Cantley, 2002). Their deficiency may compromise vascular endothelial function or osteoblast survival, exacerbating ischemia in weight-bearing bones like the femoral head. This aligns with clinical observations of reduced PC levels in steroid-induced ON and highlights their therapeutic potential (Ren et al., 2018). Conversely, sterol ester (27:1/18:3) and triacylglycerol (TAG 49:2) exhibited pro-osteonecrotic effects. TAG (49:2), enriched in glucocorticoid-associated ON, may promote lipotoxicity via adipocyte hypertrophy or impaired β-oxidation, while sterol ester accumulation could reflect dysregulated cholesterol efflux, as suggested by SREBF1 polymorphisms in ON cohorts (Lee et al., 2009; Wang et al., 2021c). Our MR study provides exploratory insights into the potential causal interplay between lipidomic profiles and ON, suggesting bidirectional associations that highlight lipidomes as both potential contributors to and consequences of bone pathology. While we applied standard sensitivity analyses, including MR-Egger intercept, Cochran’s Q, and leave-one-out analysis to evaluate heterogeneity and pleiotropy, we acknowledge that residual horizontal pleiotropy and violation of MR assumptions cannot be fully excluded. Horizontal pleiotropy, where genetic variants influence the outcome through pathways independent of the exposure, can bias causal estimates. Although the MR-Egger intercept test showed no significant evidence of directional pleiotropy and the leave-one-out and funnel plot analyses supported the consistency of our findings, these methods have limited power. Therefore, our results should be interpreted as hypothesis-generating and exploratory, rather than conclusive proof of causality, particularly given the complexity of traits like ON.

Notably, ON causally elevated 25 lipid species. This “lipid storm”, particularly esterified cholesterol and various phospholipids, could imply a disruption in cellular lipid homeostasis triggered by ON. Phosphatidylcholine and phosphatidylethanolamine are major components of cell membranes (Posor et al., 2022), and their altered levels could impact membrane integrity and function, potentially exacerbating cellular stress and contributing to the pathophysiology of ON (Yan et al., 2022). Similarly, phosphatidylinositol is involved in signaling pathways that regulate a variety of cellular processes, including apoptosis and inflammation, both of which are relevant to the progression of ON. Moreover, the consistent increase in triacylglycerol levels suggests enhanced lipid storage or impaired lipid utilization in affected tissues. This lipid accumulation could lead to lipotoxicity, further aggravating tissue damage and contributing to the progression of ON (Baek et al., 2022; Motta et al., 2022). Increased phosphatidylethanolamines (PEs) and PCs could reflect compensatory membrane repair mechanisms, while elevated esterified cholesterol points to disrupted reverse cholesterol transport a pathway central to atherosclerosis, now implicated in ON progression (Efrat et al., 2009).

Our integrative multiomics approach revealed critical biological pathways and regulatory hubs linking lipid metabolism dysregulation to ON pathogenesis. GO enrichment analysis highlighted the central role of lipid homeostasis processes, including cholesterol and triglyceride homeostasis and reverse cholesterol transport in ON development. These findings align with observations of lipid accumulation in necrotic bone and suggest that impaired lipid clearance mechanisms, rather than mere lipid overproduction, drive intraosseous ischemia (Franceschini et al., 1991; Bao et al., 2023; Tian and Yu, 2015). The enrichment of cellular components such as the Golgi apparatus and endoplasmic reticulum underscores the importance of intracellular lipid trafficking and membrane synthesis in osteocyte survival, as disrupted vesicular transport may exacerbate lipid droplet deposition and endoplasmic reticulum stress, triggering apoptosis in weight-bearing bones like the femoral head (Zhao, 2012).

KEGG pathway analysis further implicated including Notch signaling, AMPK signaling, and FA metabolism as key drivers of ON progression. Notch signaling, a well-established regulator of osteoblast-osteoclast crosstalk, may mediate lipid-induced bone remodeling defects (Gao et al., 2023). The Notch signaling pathway plays a crucial role in skeletal development and adult bone remodeling. In osteoblastic cell lines, Notch activation inhibits cell differentiation, leading to a reduction in trabecular bone mass. In adult mice, osteocyte-specific activation of Notch signaling significantly promotes bone formation, primarily by enhancing mineralization. This effect can reverse age-related bone loss and facilitate fracture healing. In contrast, inhibition of Notch signaling, such as γ-secretase deficiency, severely impairs the terminal differentiation of osteoblasts (Liu et al., 2016; Zanotti and Canalis, 2016). Concurrently, AMPK pathway inhibition (a sensor of cellular energy status) may disrupt FA oxidation, exacerbating lipid accumulation in bone marrow adipocytes (Tabe et al., 2017). These pathways collectively shape a metabolic microenvironment that influences vascular function and osteocyte viability.

PPI Network Construction and Hub Gene Identification analysis identified SCD and APOE as top hub genes, with SCD encoding stearoyl-CoA desaturase, a rate-limiting enzyme in monounsaturated FA synthesis (Ntambi et al., 2002). In our validation results, SCD expression was significantly upregulated in ON cartilage, potentially due to its role in promoting lipid droplet formation, which may render osteocytes more susceptible to lipotoxicity. Conversely, APOE dysregulation (a key cholesterol transporter) may impair reverse cholesterol transport, mirroring its role in atherosclerosis and suggesting shared mechanisms between vascular and bone pathologies (Toribio-Fernández et al., 2024). Notably, CYP7A1 downregulated in ON cartilage—highlight compromised detoxification and angiogenesis. CYP7A1 deficiency may reduce cholesterol catabolism, synergizing with lipid-driven ischemia (Moscat et al., 2003). Besides, modifications to the FADS genes impact the activity of polyunsaturated FA desaturation, which in turn alters the lipid composition in human blood and tissue. As a component of the FADS family, FADS1 regulates FA synthesis and plays a role in lipid metabolism (Koletzko et al., 2019). These findings collectively position lipid metabolism as a dynamic regulator of bone-vascular crosstalk, where hub genes orchestrate lipid handling, inflammatory responses, and osteogenic capacity.

Our findings, integrating bidirectional MR analysis, PPI network construction and hub gene identification analysis, transcriptomic and experimental analysis, provide a mechanistic framework for the pathogenesis of ON. Different lipid subspecies exhibit dual roles, either disrupting or stabilizing osteocyte lipid homeostasis. Meanwhile, necrotic bone releases lipid mediators that dysregulate systemic lipid circulation, perpetuating a self-reinforcing cycle. Targeting these lipidomes-ON-related key regulatory hubs may interrupt this cycle, offering novel therapeutic strategies for ON.

However, our study has certain limitations. We acknowledge that the lipidomic SNP-based gene annotations and transcriptomic data were derived from independent datasets. As such, the biological pathways identified from each may not fully overlap. While our multi-omics framework is designed to identify convergent biological signals, the generalizability of specific GO or KEGG terms across datasets should be interpreted with caution. It did not investigate the effects of varying exposure periods or levels of lipidomes on ON pathogenesis. The MR analysis was limited to a European population, raising questions about its generalizability to other populations. Although our MR analysis supports potential causal associations between lipid species and ON, we acknowledge that MR is not immune to residual confounding and horizontal pleiotropy. Future studies should explore these aspects to validate and extend our findings. Furthermore, we recognize the limitations of semi-quantitative IHC assessment, particularly under low magnification, and acknowledge that cellular morphology differences between ON and controls may influence staining interpretation. More precise protein quantification methods should be considered in future studies.

In conclusion, this study provides the first exploratory investigation into the bidirectional associations between 179 lipid species and ON using integrated MR and multi-omics analyses. We identified lipid species that may be protective or pathogenic in the context of ON, and observed a potential ‘lipid storm’ pattern induced by ON itself. Through PPI network construction, transcriptomic, and experimental validation, several lipid metabolism–related genes, such as APOE, PRKCA and ALK, were found to be significantly dysregulated in osteonecrotic cartilage (Figure 7). While these findings offer novel insights into lipid-bone interactions and identify promising molecular candidates, they should be considered hypothesis-generating due to limitations in MR assumptions and sample size. Future studies are warranted to validate these results in independent cohorts and mechanistic models, and to explore their translational potential for therapeutic development.