Venous ligation-mediated bone adaptation is NOS 3 dependent

Interstitial fluid flow (IFF) in bone has been hypothesized to mediate bone modeling in the absence of mechanical strain. The mechanism of this effect has not been clearly defined, though previous studies indicate that nitric oxide (NO) may play an important role in mediating IFF. In the current study, mice with a targeted disruption of the NOS 3 gene were used according to a previously established model of altered interstitial fluid flow in bone. Femoral vein ligation was performed in one limb to increase intramedullary pressure and consequently its IFF; a sham operation was performed on the contralateral limb. The mice were then hindlimb suspended to uncouple the effects of altered flow in the limb from mechanical loading. Differences in radiographic bone density and bone strength were compared for the sham and venous-ligated femurs in wild-type (WT) mice and NOS 3 knockout (KO) mice. Suspension-induced bone loss in the femurs, as evidenced by a loss in radiographic bone mineral density (BMD), was seen in both groups. Differences between sham and venous-ligated femurs were significant only for the WT mice, in which there appeared to be a protective effect of venous ligation against bone loss [-6.69% (ligated) vs. -12.36% (sham), P<0.05]. Furthermore, the difference in bone density between sham and venous-ligated femurs was muted by NOS 3 knockout, suggesting that the protective effect of venous ligation against bone loss observed in the WT group was NO dependent. The differences in relative BMD were mirrored in the mechanical testing experiments, where maximum load to fracture was significantly higher in the venous-ligated limbs relative to the sham limbs of the WT mice, but not in the NOS 3 group. Taken together, these data further support the hypothesis that fluid flow can modulate bone modeling and suggest that IFF-mediated bone adaptation is NOS 3 dependent.