Damaging Fatigue Loading Stimulates Increases in Periosteal Vascularity at Sites of Bone Formation in the Rat Ulna

Abstract

Bone formation in a variety of contexts depends on angiogenesis, however there are few reports of the vascular response to osteogenic skeletal loading. We used the rat forelimb compression model to characterize vascular changes after fatigue loading. The right forelimbs of 72 adult rats were loaded cyclically in vivo to one of four displacement levels, to produce four discrete levels of ulnar damage. Rats were euthanized 3-14 days after loading and their vasculature perfused with silicone rubber. Transverse histological sections were cut along the ulnar diaphysis. We quantified vessel number, average vessel area, total vessel area and bone area. On day 3 we observed a dramatic periosteal expansion near the ulnar midshaft, with significant increases in periosteal vascularity; total vessel area was increased 250-450% (p<0.001). Vascularity remained elevated on days 7 and 14. Vessel number and average vessel area were not correlated (p=0.09) and contributed independently to total vascular increases. Bone area was not increased on day 3, but on days 7 and 14 was increased significantly in all displacement groups (p<0.01) due to periosteal woven bone formation. Vascular and bone changes depended on longitudinal location (p<0.001), with peak increases 2 mm distal to the midshaft. Vascular and bone changes also depended on displacement level (p<0.005), with greater increases at higher levels of fatigue displacement. We conclude that skeletal fatigue loading induces a rapid increase in periosteal vascularity, followed by an increase in bone area. The angiogenic-osteogenic response is spatially coordinated and scaled to the level of the mechanical stimulus.

Comparing histological, vascular and molecular responses associated with woven and lamellar bone formation induced by mechanical loading in the rat ulna

“…In the current study, we showed an early angiogenic response following WBF loading, including upregulation of Vegf and Hif1a within 1 hour, consistent with previous reports of early Vegf expression [12-13]. By day 3, there was a significant increase in vascularity in the region where woven bone was starting to form, consistent with a previous study [32]. Because the healing response in this model is characterized by non-endochondral bone formation [28], it differs from fracture healing and DO, which include both non-endochondral and endochondral bone formation [20, 49].…”

“…Second, the number of loading cycles is variable in the WBF loading model ranging from 469 to 23463 (4-195 minutes). This variability in cycle number has been well documented in this loading model [11-12, 28, 32, 56] and was not correlated with any of our experimental outcomes. Third, one consideration in our WBF loading scenario is the formation of lamellar bone that occurs adjacent to woven bone.…”

“…Vascular perfusion was performed on a subset of rats 3 days after loading (14 rats total; n=4-5/loading group) as previously described [32]. Comparisons were made between WBF loading and rest-inserted LBF loading.…”

Hypoxia-inducible Factor-1α Protein Negatively Regulates Load-induced Bone Formation

“…This situation differs from the exogenous mechanical stimuli implemented in the current study, which are not associated with significant hypoxia or increased angiogenesis (45), but rather, are considered anabolic. On the other hand, application of much more intensive loads that induce bone fatigue with significant tissue damage is associated with increased skeletal vascularity likely due to tissue hypoxia (46,47).…”

HIF-1α regulates bone formation after osteogenic mechanical loading

“…Robust expression of HIF-1α has been observed in the inflammatory cells located in the expanded periosteal region as soon as 1 day after loading, with a peak in gene expression at day 7 [22, 25]. Additionally, woven bone formation after stress fracture is preceded by increased periosteal vascularity [25, 26] and is impaired by angiogenic inhibition [27]. In contrast to fatigue loading, mechanical loading applied near physiological strain levels for fewer cycles does not produce damage, and stimulates lamellar bone formation.…”