Morphological and histological adaptation of muscle and bone to loading induced by repetitive activation of muscle

Abstract

Muscular contraction plays a pivotal role in the mechanical environment of bone, but controlled muscular contractions are rarely used to study the response of bone to mechanical stimuli. Here, we use implantable stimulators to elicit programmed contractions of the rat tibialis anterior (TA) muscle. Miniature stimulators were implanted in Wistar rats (n = 9) to induce contraction of the left TA every 30 s for 28 days. The right limb was used as a contralateral control. Hindlimbs were imaged using microCT. Image data were used for bone measurements, and to construct a finite-element (FE) model simulation of TA forces propagating through the bone. This simulation was used to target subsequent bone histology and measurement of micromechanical properties to areas of high strain. FE mapping of simulated strains revealed peak values in the anterodistal region of the tibia (640 µε ± 30.4 µε). This region showed significant increases in cross-sectional area (28.61%, p < 0.05) and bone volume (30.29%, p < 0.05) in the stimulated limb. Histology revealed a large region of new bone, containing clusters of chondrocytes, indicative of endochondral ossification. The new bone region had a lower elastic modulus (8.8 ± 2.2 GPa) when compared with established bone (20 ± 1.4 GPa). Our study provides compelling new evidence of the interplay between muscle and bone.

Keywords: bone; loading; mechanotransduction; muscle electrical stimulation.

Figure 1.

Mid-tibial transverse MicroCT scans of the same rat hindlimb, (a) without and (b) with I₂KI-contrast enhancement. EDL, extensor digitorum longus; F, fibula; TA, tibialis anterior; TB, tibia.

Figure 2.

Volume reconstructions of tibialis anterior and extensor digitorum longus muscles in situ on the tibia of both control (left) and stimulated (right) limbs. Both stimulated muscles show a significant decrease in average muscle volume compared with the contralateral control muscle of 19% (p < 0.05) and 16% (p < 0.05), respectively. n = 6. TAc, tibialis anterior control; EDLc, extensor digitorum longus control; TAs, tibialis anterior stimulated; EDLs, extensor digitorum longus stimulated.

Figure 3.

Proportion (%) of total cross-sectional area of muscle fibre types 1, 2a and 2b within area sampled, in the stimulated and contralateral control muscles. There is a significant decrease in type 2b area (p = 0.007).

Figure 4.

Histological sections of tibialis anterior: (a,b) contralateral control muscle and (c,d) stimulated muscle. (a,c) Myofibrillar ATPase staining shows fibre type: type 1 fibres do not stain, type 2a stain darkest and 2b stain intermediate; representative fibres are labelled. (b,d) NADH dehydrogenase staining was used to stain for oxidative capacity: the darker the fibre, the more mitochondria present, and so the more oxidative the muscle. Scale bars, 100 µm.

Figure 5.

The distribution of effective strain across the tibia on the (a) medial, (b) posterior, (c) lateral and (d) anterior aspects. The highest effective strains are indicated by red regions and the lowest by blue.

Figure 6.

Regional measurements of cortical thickness along the anterior aspect of the tibia in the stimulated (broken line) and contralateral control (solid line) bones. There was a significant difference between control and stimulated bones in the distal aspect of the tibia (n = 6). Asterisks represent a highly significant difference (p < 0.001). The diagrammatic outline of the tibia represents the approximate location of the sample sites along the tibia.

Figure 7.

MicroCT data illustrating differences of bone volume and cross-sectional measurements targeted to the anterodistal region of the tibia. (a) The grey region on the three-dimensional reconstruction of the tibia highlights the region used for bone volume and cross-sectional area measurements of microCT data. (b–i) Cross-sections taken from microCT data within this region for (b–e) stimulated limb and (f–i) corresponding contralateral control limbs. (d) and (h) correspond with histological sections in figure 8a. Scale bars, 400 µm.

Figure 8.

(a) Histological cross sections of the targeted region, stained with H&E, from the tibias with the shape closest to the mean shape. (i) The contralateral control and (ii) the stimulated limb are sectioned at equivalent positions along the length of the tibia. Scale bar, 400 µm. (b) The regions of bone used for nanoindentation. White represents established bone (region A), black represents new bone (region B) and grey represents the contralateral control bone (region C). Approximate areas used for nanoindentation are outlined by dashed lines.

Figure 9.

Histological section of the stimulated tibia, showing the region of primary osteon formation. Section stained with H&E. Scale bar, 200 µm.

Figure 10.

(a) Safranin O staining of the stimulated tibia. Proteoglycans stain red; arrowheads indicate red cartilaginous regions. Scale bar, 100 µm. (b) A region with high safranin O staining, within the region of primary osteon formation on the stimulated tibia. Scale bar, 50 µm.

Figure 11.

Modulus versus displacement into surface for the anterodistal region of the contralateral control bone as determined by nanoindentation (n = 30).