DMOG pretreatment restores osteogenic–adipogenic balance and mitochondrial function in ONFH BMSCs through the HIF-1α/Homer3 pathway

Abstract

Background  Osteonecrosis of the femoral head (ONFH) is a progressive orthopedic disorder that often culminates in femoral head collapse and joint failure. Dysfunction of bone marrow mesenchymal stem cells (BMSCs), including impaired osteogenesis, enhanced adipogenesis, and mitochondrial dysfunction, has been increasingly recognized as a central driver of ONFH pathogenesis. However, the molecular mechanisms linking metabolic stress to lineage imbalance remain poorly defined. Methods  Paired BMSCs were isolated from necrotic femoral head regions (fhBMSCs) and the iliac crest (iBMSCs) of ONFH patients. Functional assays, RNA sequencing, and molecular analyses were performed to evaluate the effects of the hypoxia mimetic dimethyloxalylglycine (DMOG) on osteogenic–adipogenic balance, mitochondrial function, and senescence. Loss-of-function experiments targeting hypoxia-inducible factor-1α (HIF-1α) and Homer3 were conducted to elucidate mechanistic pathways. Results  Compared with iBMSCs, fhBMSCs exhibited impaired osteogenesis, enhanced adipogenesis, mitochondrial dysfunction, and increased senescence. DMOG pretreatment restored osteogenic differentiation, suppressed adipogenesis, improved mitochondrial dynamics, reduced oxidative stress, and enhanced bioenergetic metabolism. These protective effects were dependent on HIF-1α stabilization. Transcriptomic profiling identified Homer3 as a downstream negative regulator of HIF-1α. Homer3 was aberrantly upregulated in fhBMSCs but suppressed by DMOG, and its knockdown mimicked the effects of DMOG by promoting osteogenesis, inhibiting adipogenesis, enhancing mitophagy, and restoring mitochondrial function. Conversely, silencing HIF-1α abolished DMOG-mediated benefits and reinstated Homer3 expression.

1. Introduction

Osteonecrosis of the femoral head (ONFH) is a progressive and debilitating orthopedic disorder that frequently progresses to femoral head collapse and total hip replacement, especially in young and middle-aged patients [1]. Despite advances in surgical techniques, effective joint-preserving treatments remain limited, largely due to the incomplete understanding of disease mechanisms [2].

Bone marrow mesenchymal stem cells (BMSCs) play a pivotal role in bone homeostasis and regeneration, and their dysfunction is increasingly recognized as a major contributor to the onset and progression of ONFH [3]. BMSCs derived from ONFH patients often exhibit impaired osteogenic differentiation accompanied by enhanced adipogenesis, resulting in reduced bone formation and increased fat accumulation [4–6]. In parallel, mitochondrial dysfunction—characterized by decreased membrane potential, reduced ATP generation, excessive reactive oxygen species (ROS), and defective autophagy—further compromises BMSC differentiation capacity and lineage stability [7, 8]. However, the molecular link between metabolic stress and impaired lineage commitment in ONFH-derived BMSCs remains poorly defined.

Accumulating evidence indicates that metabolic status is a key determinant of stem cell fate. Bioenergetic and transcriptomic studies have shown that mitochondrial oxidative phosphorylation (OxPhos) is strongly activated during osteogenic differentiation of MSCs, while glycolytic activity remains largely comparable to the undifferentiated state [9, 10]. Mitochondria not only serve as the powerhouse of energy metabolism but also regulate ROS levels, phosphorylation events, and metabolic intermediates that impact transcriptional and epigenetic programs, thereby influencing lineage commitment [8, 11]. Within the pathological microenvironment of ONFH, BMSCs may be exposed to chronic ischemia, hypoxia, and glucose dysregulation, leading to mitochondrial injury and impaired autophagic activity, which further exacerbate osteogenic–adipogenic imbalance and impair bone repair [12]. Elucidating the molecular mechanisms linking mitochondrial homeostasis to BMSC differentiation abnormalities is thus critical for understanding ONFH pathogenesis and developing new therapeutic strategies.