Tendon-to-bone interface comprises a heterotypic cellular niche. The native interface is hypovascular, suggesting that the junction is physiologically hypoxic. As it bridges tendon and bone, which require different oxygen concentrations, a tight coordination of different oxygen concentrations along the junction must be considered when trying to mimic and understand biological events occurring within the tissue. Herein, an optimized in vitro co-culture model of tendon-derived cells (hTDCs) and pre-osteoblasts (pre-OBs) [1] was used to study the effect of a restricted oxygen environment on cell behavior.