Woven bone is formed in response to fatigue-induced stress fractures and is associated with increased local angiogenesis. The molecular mechanisms that regulate this woven bone formation are unknown. Our objective was to measure the temporal and spatial expression of osteo-and angiogenic genes in woven bone formation in response to increasing levels of fatigue-induced damage. We used the rat forelimb compression model to produce four discrete levels of fatigue damage in the right ulna of 115 male Fischer rats. Rats were killed at 0 (1 hr), 1, 3 and 7 days after loading. Using qRT-PCR, we quantified gene expression associated with osteogenesis (BMP2, Msx2, Runx2, Osx, BSP, Osc), cell proliferation (Hist4), and angiogenesis (VEGF, PECAM-1) from the central half of the ulna. The spatial distribution of BMP2, BSP and PCNA was assessed by immunohistochemistry or in situ hybridization in transverse histological sections 1, 4, and 7 mm distal to the ulnar mid-diaphysis. One hour after loading, BMP2 was significantly upregulated in neurovascular structures in the medial ulnar periosteum. Expression of angiogenic markers (VEGF, PECAM-1) increased significantly between Day 0 and 1 and, as with BMP2 expression, remained upregulated through Day 7. While Osx and BSP were upregulated on Day 1, the other osteogenic genes (Msx2, Runx2, Osx, BSP and Osc) were induced on Day 3 in association with the initiation of periosteal woven bone formation and continued through Day 7. The magnitude of osteogenic gene expression, particularly matrix genes (BSP, Osc) was significantly proportional the level of fatigue damage. The woven bone response to fatigue injury is remarkably similar to the "intramembranous" portion of fracture repair -rapid formation of periosteal woven bone characterized by early BMP2 expression, cell proliferation, and upregulation of osteogenic genes. We speculate that woven bone repair of fatigue damage may be an abbreviated fracture response without the requirement for endochondral repair. We conclude that bone fatigue repair is a process similar to intramembranous fracture repair characterized by increases in the expression of genes associated with angiogenesis, cell proliferation and osteoblastogenesis, and that the response from the local vasculature precedes the osteogenic response to fatigue loading.

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Diets with a high saturated fat content can produce deleterious effects on the absorption of dietary calcium and consequently an adverse effect on bone mineralization in growing animals. Hence dietary fat may have long-term consequences for skeletal health and skeletal pathologies such as osteoporosis. Whether a diet high in saturated fat has similar negative effects on adult bone, however, remains unresolved. Thus, we investigated effects of a high-fat diet on mature bone structure and mechanics. Adult (40-week-old) roosters were maintained for 20 weeks on either a diet high in saturated fat (HF) or a low-fat (LF) diet. Cortical bone samples (tarsometatarsus) were tested mechanically in three-point bending, and cancellous bone cores from the femoral condyles and tibial plateau (four sites per knee) were tested mechanically in compression. Cortical bone cross-sectional areal data were also compared among the groups, and bone mineral content (BMC) was determined (by ashing) for both cortical bone and cancellous bone samples. There were no significant high-fat diet effects on mature cortical bone mechanical properties, geometric structure, or mineral content. Diet, however, did affect cancellous bone composition. For example, LF cancellous BMC was significantly greater than HF. Mechanical properties of the cancellous bone showed similar trends such that LF cancellous bone strength was consistently greater than HF. The potential for adverse effects of a HF diet on intestinal calcium absorption in the mature animal may be more apparent in cancellous bone, with its faster rate of turnover, than in cortical bone. Changes in cancellous bone structure and mechanical properties, related to dietary saturated fats, may have implications for understanding the role of nutrition in skeletal health and prevention of pathological bone loss (osteoporosis).

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Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25 mg/kg) as well as incorporated into lipid-encapsulated αvβ3 integrin targeted nanoparticles (0.25 mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, αvβ3 integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.

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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.

No abstract