Axial strain enhances osteotomy repair with a concomitant increase in connexin43 expression

Abstract

The mechanical environment is known to influence fracture healing. We speculated that connexin43 (Cx43) gap junctions, which impact skeletal homeostasis, fracture healing and the osteogenic response to mechanical load, may play a role in mediating the response of the healing bone to mechanical strain. Here, we used an established rat fracture model, which uses a 2 mm osteotomy gap stabilized by an external fixator, to examine the impact of various cyclical axial loading protocols (2%, 10%, and 30% strain) on osteotomy healing. We examined the presence of Cx43 in the osteotomy-healing environment and assessed how mechanical strain modulates Cx43 expression patterns in the callus. We demonstrated that increased cyclical axial strain results in increased radiographic and histologic bone formation. In addition, we show by immunohistochemistry that Cx43 is abundantly expressed in the healing callus, with the expression most robust in samples exposed to increased cyclical axial strain. These data are consistent with the concept that an increase in Cx43 expression by mechanical load may be part of the mechanisms by which mechanical forces enhances fracture healing.

Figure 1

The osteotomy model, external fixator and axial-compression load rig. (a) Representative image showing the osteotomy, including the pin placement for the external fixation device. (b and c) Images of the load device, which interfaces with the external fixator and the material testing system to deliver cyclic axial compression across the osteotomy gap. (d) Image of a rat placed in a sling adapted to align the rat with the material testing system.

Figure 2

Radiographic and histologic analyses of mineralization in the callus at the 42-day time point. (a) Relative score for radiographic healing is shown for animals loaded at 2%, 10%, and 30% strain at 42-day time point (n = 3, 5, and 4, respectively). Data shown are mean ± SD. An * indicates a P < 0.05 relative to the 2% strain group. (b) Representative orthogonal radiographic images from two experimental groups at 42-day post-fracture: 2% strain (left) and 30% strain (right). (c) Relative score for histologic healing is shown for animals loaded at 2%, 10% and 30% strain at 42-day time point (n = 3, 5, and 4, respectively) (d) Representative Masson's trichrome stained section of the osteotomy gap. F, fibrous tissue; C, cartilaginous tissue; B, bony tissue. The scale bar represents 0.5 mm. The respective boxed areas are magnified in e.

Figure 3

Histomorphometric analysis of healing in the callus. (a) Measurements for total callus volume, bone volume/total volume (BV/TV), cartilage volume/total volume (CV/TV), and fibrous volume/total volume (FV/TV) within the osteotomy callus is shown for animals loaded at 2%, 10%, and 30% strain at the 10-, 21-, and 42-day time points. Data shown are mean ± SD. A indicates a P < 0.05 relative to the 2% strain group, B indicates a P < 0.05 relative to the 10 day, strain-matched data. C indicates a P < 0.05 relative to the 21-day, strain-matched data. (b) Bar charts display the composition of the healing callus at each time point and for each strain group.

Figure 4

Connexin43 expression in the callus. (a) Representative immunostaining for Cx43 in the periosteal reaction of the callus is shown for a 10-day, 10% strain animal. Arrows, periosteum; C, cortical bone; CA, cartilaginous tissue; WB, woven bone. Scale bar indicates 250 µm. (b) The mean score for the presence of Cx43 in the osteotomy callus is shown for animals loaded at 2%, 10%, and 30% strain at the 21- and 42-day time points. An * indicates a P < 0.05 relative to the 2% strain, time point-matched data. (c) Representative immunostaining for Cx43 in the rat fracture callus is shown. Note the abundant Cx43 detected in the chondrocyte and osteoblast populations of the 10% and 30% strain group are not observed in the chondrocytes in the 2% strain group. Scale bar indicates 50 µm.