Oxygen-dependent osteocyte transcriptomic adaptations occur without modulation of endothelial gene expression via osteocyte paracrine signaling

Abstract Bone regeneration remains a significant clinical challenge, requiring a deeper understanding of the cellular and molecular mechanisms underlying bone repair. Osteocytes, which represent 90%-95% of bone cells, form a connective dendritic network and play a key role in skeletal homeostasis. Embedded within the mineralized matrix, they experience unique metabolic constraints, including reduced oxygen availability. Hypoxia may, therefore, influence their function, but its impact and potential role in regulating angiogenesis during bone regeneration remain poorly understood. This study aimed to investigate (1) how hypoxia affects osteocytes and (2) their paracrine communication with endothelial cells (ECs). To model this interaction, we cultured the osteocytic cell line murine long bone osteocyte-Y4 (MLO-Y4) under normoxia (21% O₂), moderate hypoxia (5% O₂), or severe hypoxia (1% O₂) and collected the conditioned media (CM) after 48 h. Mouse ECs (1 × 104 cells/cm2) were cultured for 48 h in a 1:1 ratio of EC medium and CM from normoxic or hypoxic (5% and 1% O₂) osteocytes. Following optimization of co-culture conditions, RNA sequencing was performed to evaluate osteocyte and ECs’ gene expression changes. Our findings indicate that hypoxia contributes to the development of the osteocyte dendritic network and upregulates neurogenesis-associated genes. Moreover, oxygen levels differentially influence osteocyte behavior by modulating their hypoxic response and metabolism. Indeed, severe hypoxia (1% O₂) induces hypoxia-responsive genes and glycolysis more strongly than moderate hypoxia (5% O₂), resulting in distinct metabolic and transcriptional profiles. However, osteocyte-derived factors did not significantly alter gene expression in ECs, suggesting that paracrine signaling, which is possibly not modulated by hypoxia, is not the principal mode of communication involved. Further studies are required to clarify the mechanisms by which oxygen levels shape osteocyte physiology.