Functional adaptation to mechanical loading in both cortical and cancellous bone is controlled locally and is confined to the loaded bones

Abstract

In order to validate whether bones' functional adaptation to mechanical loading is a local phenomenon, we randomly assigned 21 female C57BL/6 mice at 19 weeks of age to one of three equal numbered groups. All groups were treated with isoflurane anesthesia three times a week for 2 weeks (approximately 7 min/day). During each anaesthetic period, the right tibiae/fibulae in the DYNAMIC+STATIC group were subjected to a peak dynamic load of 11.5 N (40 cycles with 10-s intervals between cycles) superimposed upon a static "pre-load" of 2.0 N. This total load of 13.5 N engendered peak longitudinal strains of approximately 1400 microstrain on the medial surface of the tibia at a middle/proximal site. The right tibiae/fibulae in the STATIC group received the static "pre-load" alone while the NOLOAD group received no artificial loading. After 2 weeks, the animals were sacrificed and both tibiae, fibulae, femora, ulnae and radii analyzed by three-dimensional high-resolution (5 mum) micro-computed tomography (microCT). In the DYNAMIC+STATIC group, the proximal trabecular percent bone volume and cortical bone volume at the proximal and middle levels of the right tibiae as well as the cortical bone volume at the middle level of the right fibulae were markedly greater than the left. In contrast, the left bones in the DYNAMIC+STATIC group showed no differences compared to the left or right bones in the NOLOAD or STATIC group. These microCT data were confirmed by two-dimensional examination of fluorochrome labels in bone sections which showed the predominantly woven nature of the new bone formed in the loaded bones. We conclude that the adaptive response in both cortical and trabecular regions of bones subjected to short periods of dynamic loading, even when this response is sufficiently vigorous to stimulate woven bone formation, is confined to the loaded bones and does not involve changes in other bones that are adjacent, contra-lateral or remote to them.

Fig. 1

Direction of mechanical loading in the tibia/fibula and representative transverse μCT images at the analyzed sites in the tibia, fibula, femur, ulna and radius of a 21-week-old female C57BL/6 mouse. In the tibia, proximal (25% of the bone's length from its proximal end), proximal/middle (37%), middle (50%) and distal (75%) sites in cortical bone and two sites 0.01–0.25 mm (containing primary spongiosa) and 0.25–1.25 mm (secondary spongiosa) distal to growth plate in trabecular bone were analyzed. In the fibula, femur, ulna and radius, the middle (50%) sites in cortical bones were analyzed.

Fig. 2

Relative values, analyzed by μCT and histomorphometry, of the left and right bones in the NOLOAD, STATIC and DYNAMIC + STATIC groups compared to the left bones in the NOLOAD group. L = left, R = right. (A) Cortical bone volume analyzed by μCT at the proximal (25% of the bone's length from its proximal end), proximal/middle (37%), middle (50%) and distal (75%) sites of the tibia. (B) Periosteal labels and inter-label bone area, analyzed by histomorphometry, normalized by total cortical bone area at the proximal, proximal/middle, middle and distal sites of the tibia. (C) Endosteal labels and inter-label bone area, analyzed by histomorphometry, normalized by total cortical bone area at the proximal, proximal/middle, middle and distal sites of the tibia. (D) Trabecular percent bone volume analyzed by μCT at two sites 0.01–0.25 mm (containing primary spongiosa) and 0.25–1.25 mm (secondary spongiosa) distal to the growth plate in the proximal tibia. (E) Cortical bone volume analyzed by μCT at the middle (50%) site of the fibula, femur, ulna and radius. Data are the mean ± SE (n = 6–7). ⁎p < 0.05 versus all other five values by one-way ANOVA followed by a post hoc Bonferroni or Dunnett T3 test.

Fig. 3

Representative transverse fluorochrome-labelled images. (A) Cortical bone at the proximal (25% of the bone's length from its proximal end), proximal/middle (37%), middle (50%) and distal (75%) sites of the tibia. (B) Trabecular bone at the site 0.25 mm distal to the growth plate in the proximal tibia. (C) Cortical bone at the middle (50%) site of the fibula. Green: calcein label injected on the first day of loading (day 1). Red: alizarin label injected on the last day of loading (day 12).