Non-traumatic osteonecrosis of the femoral head (NONFH) is a prevalent and challenging orthopedic condition that primarily affects young and middle-aged individuals, with early diagnosis and treatment being exceedingly difficult (Hines et al., 2021). This disease is characterized by the collapse of the femoral head and degenerative changes in the hip joint, severely compromising patients' quality of life (Lee et al., 2010). NONFH has a high incidence rate, with an estimated 10,000–20,000 new cases annually in the United States and 100,000 to 200,000 new cases reported each year in China (Zhao et al., 2015, Xu et al., 2020). However, during the early stages of NONFH, patients typically exhibit no significant symptoms beyond occasional hip joint pain or discomfort. The absence of noticeable X-ray changes in the early stages fails to raise sufficient alarm, and by the time of diagnosis, the disease is often irreversible.

Current treatments for NONFH include conservative management (e.g., weight-bearing restriction and physical therapy), pharmacotherapy (e.g., anticoagulants, vasodilators, and lipid-lowering drugs), and surgical interventions (e.g., core decompression, vascularized bone grafting, and total hip arthroplasty) (Xu et al., 2020, Migliorini et al., 2023, Migliorini et al., 2021, Sadile et al., 2016, Migliorini et al., 2022). However, conservative approaches offer limited symptom relief, pharmacological treatments often fall short of reversing disease progression, and surgical options face challenges such as technical complexity and finite prosthesis lifespan (Quaranta et al., 2021). Each strategy has limitations, and younger patients may require multiple revisions, highlighting the need for more effective and durable treatments. If untreated, patients with NONFH are at risk of inevitable femoral head collapse, progressing to the end stage of the disease, which may necessitate total hip arthroplasty (THA). The prostheses used in THA have a limited lifespan, and for young and middle-aged patients, revision surgery will be required once the prosthesis wears out. Post-THA patients also face lifestyle restrictions, imposing significant psychological stress and a substantial economic burden on both the individuals and society (Scott et al., 2022). The difficulty in early diagnosis and treatment of NONFH is primarily due to the unclear pathophysiological mechanisms. To date, hypotheses regarding the pathogenesis of NONFH mainly include an imbalance between osteogenic and adipogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and impairment of vascular endothelial cells and blood supply in the femoral head (Li et al., 2020a). BMSCs, which possess multilineage differentiation potential, are considered the progenitors of osteogenic cells in the femoral head and play a crucial role in bone tissue growth, regeneration, and repair (Chu et al., 2020, Ma et al., 2023). It has been reported that the differentiation of BMSCs into osteogenic cells is significantly abnormal in the femoral heads of NONFH patients (Li et al., 2020b). Additionally, in this disease, the vasculogenic cell function of vascular endothelial cells is reduced, their migration capacity is weakened, and the secretion of VEGF protein is diminished, leading to sparse capillaries in the femoral head, a disorganized fibrinolytic system, thrombosis, and a severe reduction in blood supply to the trabecular bone (Hu et al., 2021a). Therefore, studying the reasons for the decrease in osteogenesis and angiogenesis within the femoral head of NONFH patients is of great significance and will aid in the prevention and early treatment of NONFH.

Exosomes are nanoscale vesicles, 40–150 nm in diameter, formed through a continuous process of internalization-fusion-secretion (Selvadoss et al., 2024, Tan et al., 2024). They possess membranes very similar to their parent cells and contain a wealth of proteins, lipids, and non-coding RNAs (Zhao and Huang, 2024). Almost all cells in the human body can produce and secrete exosomes, which are widely present in various extracellular fluids. Once exosomes enter cells, they can mediate intercellular signal transduction and play a regulatory role (Zhang et al., 2024). Among these components, MicroRNAs (miRNAs) are of particular interest. miRNAs are small, non - coding RNAs that can regulate gene expression at the post - transcriptional level by binding to the 3′-untranslated region (3′-UTR) of target mRNAs, leading to mRNA degradation or translational repression. In the context of exosomes, miRNAs can be transferred between cells, mediating intercellular communication and influencing various biological processes. The therapeutic role of miRNAs in orthopedics has been well documented, including osteoarthritis, tendon homeostasis, rheumatoid arthritis and tendon injuries (Giordano et al., 2020, Oliviero et al., 2019, Gargano et al., 2021, Gargano et al., 2022, Gargano et al., 2023). Regarding NONFH, previous studies have shown that miRNAs are involved in its regulation and treatment. For example, miR-100b-5p has been reported to promote osteogenic differentiation of BMSCs and may be a potential therapeutic target for NONFH (Yang et al., 2022). While exosomal miRNAs (e.g., miR-100b-5p, miR-206) have been implicated in osteoporosis and osteoarthritis, their roles in NONFH remain underexplored. Unlike osteoporosis, where exosomal miRNAs primarily regulate systemic bone loss, NONFH involves localized femoral head necrosis driven by combined osteogenic/adipogenic imbalance and vascular insufficiency. Our study is the first to identify exosomal miR-214–3p as a key mediator linking these two pathological processes in NONFH, filling a critical knowledge gap. Notably, miR-214–3p has been previously implicated in bone homeostasis: studies have shown it inhibits osteogenic differentiation of BMSCs by targeting β-catenin and RUNX2 in osteoporosis models (Tang et al., 2024), and its upregulation correlates with impaired bone formation in diabetic bone loss (Wang et al., 2019a). Additionally, miR-214–3p has been reported to suppress angiogenesis by downregulating VEGF signaling in endothelial cells (Xiao et al., 2021), making it a strong candidate for mediating the dual defects of osteogenesis and angiogenesis in NONFH. However, the role of miR-214–3p in NONFH-derived exosomes and their detailed molecular mechanisms remain unclear. Exosomes have been reported for use in the treatment of NONFH, but to date, no detailed studies have been conducted on the role and detailed molecular mechanisms of exosomes released from the necrotic bone tissue in this disease (Yang et al., 2022, Zhu et al., 2020). Therefore, studying exosomes released from the necrotic bone tissue of NONFH patients will help reveal the reasons for the failure of BMSCs transplantation and the failure of autologous BMSCs repair.

Our study addresses two central questions: First, how do exosomes derived from NONFH influence the differentiation and angiogenesis of BMSCs? Second, what are the underlying molecular mechanisms by which these exosomes exert their effects? Drawing on existing research and our preliminary findings, we hypothesize that NONFH-derived exosomes carry specific molecules, which inhibit the osteogenic differentiation of BMSCs and impede angiogenesis. We propose that these actions play a significant role in the pathogenesis of NONFH. In this study, we extracted NONFH exosomes to explore their impact on BMSCs, HUVECs, and rats, revealing the role of NONFH exosomes in the pathogenesis and progression of the disease, elucidating the potential reasons for the failure of BMSCs transplantation and autologous BMSCs repair. Furthermore, we analyzed the potential mechanisms by which NONFH exosomes affect hBMSCs, HUVECs, and rat femurs based on the GEO database analysis. This study focuses on the role of NONFH - derived exosomes in the pathogenesis of the disease, which has multiple clinical significances. Firstly, by in - depth studying the roles and mechanisms of exosomes and their carried MicroRNAs in NONFH, we are expected to discover new biomarkers, which will contribute to the early diagnosis of NONFH, thus achieving early detection and treatment of the disease. Secondly, this study may provide new targets and strategies for the treatment of NONFH. For example, if the functions of specific MicroRNAs in exosomes can be clarified, the disease process can be intervened by regulating the expression or activity of these MicroRNAs. In addition, the research results may also provide a reference for evaluating the prognosis of NONFH patients, helping to formulate personalized treatment plans, improve treatment effects, and enhance the quality of life of patients.