The mouse fibula as a suitable bone for the study of functional adaptation to mechanical loading

Abstract

Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation. The right tibiae/fibulae in C57BL/6 mice were subjected to a single period of axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle; approximately 7 min/day, 3 alternate days/week, 2 weeks). The left tibiae/fibulae were used as non-loaded, internal controls. Both left and right fibulae and tibiae were analyzed by micro-computed tomography at the levels of the mid-shaft of the fibula and 25% from its proximal and distal ends. We also investigated the effects of intermittent parathyroid hormone (iPTH) on the (re)modelling response to 2-weeks of loading and the effect of 2-consecutive days of loading on osteocytes' sclerostin expression. These in vivo experiments confirmed that the fibula showed similar loading-related (re)modelling responses to those previously documented in the tibia and similar synergistic increases in osteogenesis between loading and iPTH. The numbers of sclerostin-positive osteocytes at the proximal and middle fibulae were markedly decreased by loading. Collectively, these data suggest that the mouse fibula, as well as the tibia and ulna, is a useful bone in which to assess bone cells' early responses to mechanical loading and the adaptive (re)modelling that this engenders.

Fig. 1

Direction of mechanical loading in the right tibia/fibula and representative transverse μCT images of the fibula and tibia at the analyzed sites (25% proximal, middle and 25% distal sites of the fibula's length) in a 19 week old female C57BL/6 mouse.

Fig. 2

Effects of 2-weeks of mechanical loading with a peak load of 13.5 N on the fibula and tibia in 19 week old female C57BL/6 mice. A: Load-induced percentage increases ([right loaded − left control] × 100 / left control) in cortical bone volume of the fibula and tibia. Mean ± S.E. (n = 5). ⁎p < 0.05 by paired t-test between left control and right loaded. ^#p < 0.05 by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test among different sites. B: Representative transverse μCT images of the fibula and tibia. C: Representative transverse fluorochrome labelled images of the fibula. Green: calcein labels injected on the first and last days of loading (days 1 and 12).

Fig. 3

Effects of 2-weeks of mechanical loading with a peak load of 12.0 or 15.8 N, which produced a similar level of strain measured at the medial surface of the tibia, on the fibula and tibia in 17 week old female C57BL/6 mice treated with vehicle or iPTH (1–34) for 6 weeks, respectively. A: Load-induced percentage increases ([right loaded − left control] × 100 / left control) in cortical bone volume of the fibula and tibia. Mean ± S.E. (n = 5). ⁎p < 0.05 by paired t-test between left control and right loaded. ^#p < 0.05 by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test among different sites. B: Representative transverse μCT images of the fibula and tibia in the mice treated with iPTH (1–34). C: Representative transverse fluorochrome labelled images of the fibula in the mice treated with iPTH (1–34). Green: calcein labels injected on the first days of iPTH (1–34) treatment (day 1) and loading (day 29). Red: alizarin label injected on the last day of loading (day 41).

Fig. 4

Effect of 2-consecutive days of mechanical loading with a peak load of 13.5 N on osteocytes' sclerostin expression in the fibula of 19 week old female C57BL/6 mice. A: Representative transverse sclerostin-immunostained images 24 h after the last loading. B: Negative control of sclerostin-immunostained image. C: Mechanical load-induced percentage decreases in sclerostin-positive osteocytes ([right loaded − left control] × 100 / left control). Mean ± S.E. (n = 6). ⁎p < 0.05 by paired t-test between left control and right loaded.