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 killed 3-14 days after loading, and their vasculature was 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.

Figure 1

Photomicrographs of transverse histological sections from a control (left) ulna at five sites along the ulnar length. Perfused vessels were easily visualized as opaque areas within the thin periosteal layer, as well as within bone and muscle. (stained with toluidine blue; scale bar = 500μm) P4: 4 mm proximal to the midpoint; P2: 2 mm proximal to the midpoint; M: midpoint; D2: 2 mm distal to the midpoint; D4: 4 mm distal to the midpoint.

Figure 2

Photomicrographs of transverse histological sections from six loaded (right) and one control (left) ulnae taken 2 mm distal to the midpoint (D2) (orientation same as in Figure 1). (a–f) Low power images of representative samples from the lowest (30%) and highest (85%) displacement groups 3, 7 and 14 days after fatigue loading (10X objective; scale bar = 200 μm). (g–i) Higher power images of ulnae on day 3 (40X objective; scale bar = 50 μm). Loading caused a dramatic expansion of the periosteal and sub-periosteal tissue layers by day 3 for all displacement groups, with greater expansion observed in the higher displacement groups; note that the muscle [M] has been displaced away from the bone [B] in the loaded ulnae compared to control. The number and size of periosteal vessels were greatly increased in loaded ulnae. On day 3, a clear demarcation is seen at higher power (h,i) between the sub-periosteal layer (tissue between the original bone and the periosteal margin [P]) and the periosteal layer (which we define as the tissue between the periosteal margin and the muscle). The sub-periosteal layer is filled with numerous plump cuboidal cells surrounding the blood vessels, and small buds of newly formed bone (pink) are seen; the periosteal layer has a looser, fibrovascular appearance. By day 7, the sub-periosteal layer has transformed to woven bone and the periosteal layer is reduced in size. Isolated regions of chondroid bone (d, *) were observed in the 85% displacement group at this timepoint. By day 14, the woven bone layer has consolidated and the periosteal layer is further reduced. On days 7 and 14, the major site of new vessels is in channels in the woven bone, whereas at day 3 the new vessels are not yet enveloped in bone. (stained with hematoxylin and eosin)

Figure 2

Photomicrographs of transverse histological sections from six loaded (right) and one control (left) ulnae taken 2 mm distal to the midpoint (D2) (orientation same as in Figure 1). (a–f) Low power images of representative samples from the lowest (30%) and highest (85%) displacement groups 3, 7 and 14 days after fatigue loading (10X objective; scale bar = 200 μm). (g–i) Higher power images of ulnae on day 3 (40X objective; scale bar = 50 μm). Loading caused a dramatic expansion of the periosteal and sub-periosteal tissue layers by day 3 for all displacement groups, with greater expansion observed in the higher displacement groups; note that the muscle [M] has been displaced away from the bone [B] in the loaded ulnae compared to control. The number and size of periosteal vessels were greatly increased in loaded ulnae. On day 3, a clear demarcation is seen at higher power (h,i) between the sub-periosteal layer (tissue between the original bone and the periosteal margin [P]) and the periosteal layer (which we define as the tissue between the periosteal margin and the muscle). The sub-periosteal layer is filled with numerous plump cuboidal cells surrounding the blood vessels, and small buds of newly formed bone (pink) are seen; the periosteal layer has a looser, fibrovascular appearance. By day 7, the sub-periosteal layer has transformed to woven bone and the periosteal layer is reduced in size. Isolated regions of chondroid bone (d, *) were observed in the 85% displacement group at this timepoint. By day 14, the woven bone layer has consolidated and the periosteal layer is further reduced. On days 7 and 14, the major site of new vessels is in channels in the woven bone, whereas at day 3 the new vessels are not yet enveloped in bone. (stained with hematoxylin and eosin)

Figure 3

Increases in vessel parameters and bone area depended significantly on longitudinal location. Values represent the overall mean (± SD) from all 12 loaded groups (N = 67–70) and are representative of the pattern seen in each of the groups. With the exception of bone area at P4, significant increases were observed over the entire 8 mm region of the central ulna (P4 to D4). Vessel number, total vessel area and bone area each increased in a similar pattern, with peaks occurring at section D2. Average vessel area increased in a slightly different pattern, with a broad peak from P2 to D2. (Section locations defined in Figure 1.)

Figure 4

Increases in vessel parameters and bone area depended significantly on fatigue displacement. Values are mean (± SD) percent increases in the loaded ulnae versus the control group at section D2. Note the large magnitude of the vascular and osseous response, with mean peak increases of approximately 275% in vessel number, 110% in average vessel area, 500% in total vessel area, and 100% in bone area. a: different from 30% group at same time (p < 0.05); b: different from 45% group at same time (p < 0.05); c: different from 65% group at same time (p < 0.05)