Culture of Saos-2 cells under hypoxic conditions stimulates rapid differentiation to an osteocyte-like stage

The intracellular infection of osteocytes represents a clinically important aspect of osteomyelitis. However, few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast differentiation through to a mature osteocyte-like stage. Culture under osteogenic conditions in a standard 5% CO2 and normoxic (21% O2) atmosphere results in reproducible mineralisation and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation of Saos-2 cells. Cells cultured in a 5% CO2, 1% O2 atmosphere exhibited accelerated deposition of mineral, reaching near saturation by 14 days as demonstrated with the Alizarin Red and Von Kossa staining. The gene expression of the major hypoxia-induced transcription factor HIF1α and the key osteogenic transcription factor RUNX2 were both elevated under 1% O2. Early (COLA1, MEPE) and mature (PHEX, DMP1 and SOST) osteocyte markers were also upregulated earlier under hypoxic compared to normoxic growth conditions. Thus, culture under low oxygen accelerates key markers of osteocyte differentiation, resulting in a useful human osteocyte-like in vitro cell model within 14 days. ### Competing Interest Statement The authors have declared no competing interest.