Osteocyte apoptosis is mechanically regulated and induces angiogenesis in vitro

Osteocyte apoptosis, associated with reduced interstitial fluid flow, precedes osteoclast precursor recruitment and may aid in the delivery of osteoclast precursors to the remodeling site by promoting angiogenesis. To test the association between fluid flow and osteocyte apoptosis, osteocyte-like MLO-Y4 cells were subjected to either oscillatory fluid flow (10 dynes/cm², 1 Hz) or no flow conditions with or without TNF-α treatment to induce osteocyte apoptosis chemically. Flow protected osteocytes from apoptosis regardless of whether they were treated with TNF-α (p < 0.001) or not (p < 0.05). TNF-α-induced apoptotic and nonapoptotic osteocyte conditioned media were used to study the effect of osteocyte apoptosis on angiogenesis. Apoptotic osteocyte conditioned media caused more endothelial cell proliferation (p < 0.05) and migration (p < 0.05), and tubule networks with longer (p < 0.01) and more (p < 0.001) branches than nonapoptotic osteocyte conditioned media. Apoptotic osteocyte conditioned media contained more vascular endothelial growth factor (VEGF) than nonapoptotic osteocyte conditioned media (p < 0.05). VEGF concentrations found in apoptotic osteocyte conditioned media formed endothelial tubule networks with longer (p < 0.05) and more (p < 0.02) branches than VEGF concentrations in nonapoptotic osteocyte conditioned media. Blocking VEGF in apoptotic osteocyte conditioned media abolished tubule formation effects (p < 0.001). Our results suggest that osteocyte apoptosis is flow-regulated and promotes angiogenesis in a VEGF-mediated manner.