In vivo static creep loading of the rat forelimb reduces ulnar structural properties at time-zero and induces damage-dependent woven bone formation

Abstract

Periosteal woven bone forms in response to stress fractures and pathological overload. The mechanical factors that regulate woven bone formation are poorly understood. Fatigue loading of the rat ulna triggers a woven bone response in proportion to the level of applied fatigue displacement. However, because fatigue produces damage by application of cyclic loading it is unclear if the osteogenic response is due to bone damage (injury response) or dynamic strain (adaptive response). Creep loading, in contrast to fatigue, involves application of a static force. Our objectives were to use static creep loading of the rat forelimb to produce discrete levels of ulnar damage, and subsequently to determine the bone response over time. We hypothesized that 1) increases in applied displacement during loading correspond to ulnae with increased crack number, length and extent, as well as decreased mechanical properties; and 2) in vivo creep loading stimulates a damage-dependent dose-response in periosteal woven bone formation. Creep loading of the rat forelimb to progressive levels of sub-fracture displacement led to progressive bone damage (cracks) and loss of whole-bone mechanical properties (especially stiffness) at time-zero. For example, loading to 60% of fracture displacement caused a 60% loss of ulnar stiffness and a 25% loss of strength. Survival experiments showed that woven bone formed in a dose-dependent manner, with greater amounts of woven bone in ulnae that were loaded to higher displacements. Furthermore, after 14 days the mechanical properties of the loaded limb were equal or superior to control, indicating functional repair of the initial damage. We conclude that bone damage created without dynamic strain triggers a woven bone response, and thus infer that the woven bone response reported after fatigue loading and in stress fractures is in large part a response to bone damage.

Figure 1

Representative creep curves as a function of normalized time. The force is ramped up within 0.25 seconds and held constant for the duration of the test. Displacement changes after 0.25 seconds are considered to be creep displacement. The test shown here ended when fracture occurred (at 6.9 min). The relative actuator position tracks the displacement through three stages of creep [4, 5, 7]. The primary stage has an initial rapid increase in creep displacement followed by gradually decreasing slope. The secondary creep stage follows with a relatively constant creep rate. The tertiary creep stage begins with a rapid acceleration in creep rate leading to fracture. The average displacement to fracture was 2.32 ± 0.46 mm relative to the displacement at 5 seconds. (Five seconds was used as a reference time to match the reference time used in our previous fatigue studies [29]. Displacement in the first 5 seconds of the test is attributed to elastic deformation of the forelimb and soft tissue creep.) In subsequent sub-fracture experiments, actuator displacement was monitored and stopped at a percentage of the average fracture displacement.

Figure 2

Increasing displacement caused progressive loss of time-zero bone stiffness. (The percent change in stiffness for loaded (R) versus control (L) limbs was calculated using (R-L)/ L*100.) For all displacement groups beyond 20%, loaded ulnae were significantly less stiff than controls. Beyond 60% displacement there were no further reductions in stiffness. (P<0.05: * vs. control; a vs. 20%; b vs. 30%, 40% and 50%)

Figure 3

A clear dose-response was seen in the total amount of woven bone formed after creep loading. (A) Histological and microCT images illustrate increasing amounts of periosteal woven bone formation 1 mm distal to the midpoint of the bone for low (20%), medium (40%) and high (80%) displacement groups. (B) Quantification of woven bone area (totaled from all histomorphometry slides at five locations) demonstrates significant differences between displacement groups. (P<0.05: * vs. control; a vs. 20%; b vs. 40%)

Figure 4

The magnitude and type of bone formed in response to creep loading varies longitudinally, similar to the pattern observed after fatigue loading [28]. (Data from 40% displacement group shown; other groups had similar distributions) (A) The percentage change in BMC peaks at the midpoint (MP) and decreases both in the proximal and distal directions away from the midpoint. (B) Histological images illustrate labeled surfaces at five locations along the ulna. Woven bone is greatest 1 mm distal to the midpoint (D1) on the medial side of the bone. (P<0.05: * vs. control; a vs. D8; b vs. D6; c vs. D4; d vs. D2; e vs. MP; f vs. P2)

Figure 5

Initial significant decreases in stiffness and ultimate force were recovered 14 days after loading. Percentage change is based on comparison to contralateral controls. There were significant increases over the 14 day recovery period in both of these parameters. The ultimate force of the loaded (Right) limb was significantly higher compared to control (Left) at 14 days post loading. (*P<0.05 vs. control)