VASCULAR RESPONSE TO BONE INJURY: NOS-MEDIATED VASODILATION AND INCREASED BLOOD FLOW RATE FOLLOWING STRESS FRACTURE IN RATS

Summary Statement Bone stress fracture triggers a rapid increase in blood flow in association with mast cell production of inducible nitric oxide synthase (iNOS). NOS inhibition blocks the increase in blood flow and reduces woven bone formation needed for stress fracture healing. Introduction Vascular-bone interactions are critical in skeletal development and fracture healing. We recently showed that angiogenesis is required for stress fracture healing. However, the changes in vascularity that occur in the first 72 hours after stress fracture can not be explained by angiogenesis. Here, we evaulated early changes in blood flow and vasodilation after either damaging (stress fracture) or non-damaging mechanical loading in rats. Methods The right forelimbs of adult rats were subjected to cyclic axial compression in vivo. We used two established protocols: damaging loading that creates a stress fracture and leads to woven bone formation (WBF loading), or non-damaging loading that stimulates lamellar bone formation (LBF loading). PET imaging was used to evaluate blood flow and fluoride kinetics based on uptake of 15 O water and 18 F fluoride radioisotopes, respectively, at the site of bone formation. To quantify vasodilation, the area of the anterior interosseous artery was measured. Inducible nitric oxide synthase (iNOS) expression was evaluated by immunostaining. Finally, NO production was impaired by administration of L-NAME ( N ω -nitro-L-arginine methyl ester), a NOS inhibitor. Results PET Imaging: Damaging WBF loading induced early and persistent increases in blood flow. Blood flow rate was increased ∼30% at 4 hours through 14 days in WBF loaded limbs. Fluoride uptake peaked 7 days after WBF loading, then declined from 7 to 14 days, consistent with the dynamics of woven bone formation described previously. Non-damaging LBF loading did not affect blood flow or fluoride kinetics. Histology: WBF loaded limbs had significantly increased arterial area (+50%) compared to non-loaded limbs at days 1 and 3, with return to normal by day 7. LBF loading did not affect arterial area. Since mast cells are a possible effector of vasodilation, mast cell infiltration and iNOS expression were quantified following loading. iNOS+ mast cells in WBF-loaded limbs were significantly increased on days 1 and 3, with return to normal by day 7. LBF loading was not associated with increases in iNOS+ mast cells. NOS Inhibition: L-NAME blocked the expression of iNOS in mast cells following WBF loading. Additionally, L-NAME treatment abolished the increase in blood flow rate at days 1 and 3, and diminished fluoride uptake at day 3. Finally, L-NAME treatment decreased woven bone formation, with significant decreases in woven bone volume (−27%) and BMD (−26%), compared to vehicle controls. Discussion/Conclusion Damaging loading produces a stress fracture and leads to woven bone formation (WBF). Prior to bone formation, there is a rapid increase in blood flow rate in association with vasodilation and infiltration of iNOS+ mast cells in the expanded periosteum. Inhibition of NOS blocks the increase in blood flow rate, and ultimately impairs woven bone formation. In contrast, non-damaging (LBF) loading does not affect blood flow rate, vasodilation, or iNOS expression in mast cells. Thus, the vascular response after stress fracture involves an early increase in blood flow by vasodilation, followed by angiogenesis to maintain increased blood flow. Disruption of either response affects subsequent bone formation during stress fracture healing.