Discussion
In osteocyte cells subjected to the double stresses of glucocorticoid administration and a hypoxic environment osteocytic cell death was mediated via mitochondria. In contrast, MSC subjected to the same stressors showed preservation of mitochondrial function and reduced oxidative stress. Accordingly, even under conditions sufficiently stressful to cause the osteocytic cell death in vivo, it was thought that the function of MSC could be preserved, suggesting that in the case of osteonecrosis preventative and therapeutic strategies incorporating their intraosseous implantation may be promising.
Methods
Mouse bone cells (MLO-Y4) and bone-marrow derived mouse MSCs were exposed to dexamethasone (Dex), hypoxia of 1% oxygen or both in vitro. Mitochondrial membrane potentials were estimated by mitochondria labeling with a cell-permeant probe (Mito tracker red); expression of these apoptosis-inducing molecules, oxidative stress marker (8-hydroxy-2'-deoxyguanosine), caspase-3, -9, and two apoptosis-inhibiting molecules, energy-producing ATP synthase (ATP5A) and X-linked inhibitor of apoptosis protein (XIAP), were analyzed by both immunofluorescence and western blot.
Results
With exposure to either dexamethasone or hypoxia, MLO-Y4 showed reduced mitochondrial membrane potential, ATP5A and upregulation of 8-OHdG, cleaved caspases and XIAP. Those changes were significantly enhanced by treatment with dexamethasone plus hypoxia. In MSCs, however, mitochondrial membrane potentials were preserved, while no significant changes in the pro-apoptosis or anti-apoptosis molecules analyzed were found even with exposure to both dexamethasone and hypoxia. No such effects induced by treatment with dexamethasone, hypoxia, or both were demonstrated in MSCs at all.