Micro-computed tomography assessment of the progression of fracture healing in mice.

The mouse fracture model is ideal for research into the pathways of healing because of the availability of genetic and transgenic mice and the ability to create cell-specific genetic mutations. While biomechanical tests and histology are available to assess callus integrity and tissue differentiation, respectively, micro-computed tomography (µCT) analysis has increasingly been utilized in fracture studies because it is non-destructive and provides descriptions of the structural and compositional properties of the callus. However, the dynamic changes of µCT properties that occur during healing are not well defined. Thus, the purpose of this study was to determine which µCT properties change with the progression of fracture repair and converge to values similar to unfractured bone in the mouse femur fracture model. A unilateral femur fracture was performed in C57BL/6 mice and intramedullary fixation performed. Fractured and un-fractured contralateral specimens were harvested from groups of mice between 2 and 12 weeks post-fracture. Parameters describing callus based on µCT were obtained, including polar moment of inertia (J), bending moment of inertia (I), total volume (TV), tissue mineral density (TMD), total bone volume fraction (BV/TV), and volumetric bone mineral density (vBMD). For comparison, plain radiographs were used to measure the callus diameter (D) and area (A); and biomechanical properties were evaluated using either three-point bending or torsion. The µCT parameters J, I, TV, and TMD converged toward their respective values of the un-fractured femurs over time, although significant differences existed between the two sides at every time point evaluated (p<0.05). Radiograph measurement D changed with repair progression in similar manner to TV. In contrast, BV/TV and BMD increased and decreased over time with statistical differences between callus and un-fractured bone occurring sporadically. Similarly, none of the biomechanical properties were found to distinguish consistently between the fractured and un-fractured femur. Micro-CT parameters assessing callus structure and size (J, I, and TV) were more sensitive to changes in callus over time post-fracture than those assessing callus substance (TMD, BV/TV, and BMD). Sample size estimates based on these results indicate that utilization of µCT requires fewer animals than biomechanics and thus is more practical for evaluating the healing femur in the mouse fracture model.

Fracture healing in protease-activated receptor-2 deficient mice.

Protease-activated receptor-2 (PAR-2) provides an important link between extracellular proteases and the cellular initiation of inflammatory responses. The effect of PAR-2 on fracture healing is unknown. This study investigates the in vivo effect of PAR-2 deletion on fracture healing by assessing differences between wild-type (PAR-2(+/+)) and knock-out (PAR-2(-/-)) mice. Unilateral mid-shaft femur fractures were created in 34 PAR-2(+/+) and 28 PAR-2(-/-) mice after intramedullary fixation. Histologic assessments were made at 1, 2, and 4 weeks post-fracture (wpf), and radiographic (plain radiographs, micro-computed tomography (µCT)) and biomechanical (torsion testing) assessments were made at 7 and 10 wpf. Both the fractured and un-fractured contralateral femur specimens were evaluated. Polar moment of inertia (pMOI), tissue mineral density (TMD), bone volume fraction (BV/TV) were determined from µCT images, and callus diameter was determined from plain radiographs. Statistically significant differences in callus morphology as assessed by µCT were found between PAR-2(-/-) and PAR-2(+/+) mice at both 7 and 10 wpf. However, no significant histologic, plain radiographic, or biomechanical differences were found between the genotypes. The loss of PAR-2 was found to alter callus morphology as assessed by µCT but was not found to otherwise effect fracture healing in young mice.

Increasing duration of type 1 diabetes perturbs the strength-structure relationship and increases brittleness of bone.

Type 1 diabetes (T1DM) increases the likelihood of a fracture. Despite serious complications in the healing of fractures among those with diabetes, the underlying causes are not delineated for the effect of diabetes on the fracture resistance of bone. Therefore, in a mouse model of T1DM, we have investigated the possibility that a prolonged state of diabetes perturbs the relationship between bone strength and structure (i.e., affects tissue properties). At 10, 15, and 18 weeks following injection of streptozotocin to induce diabetes, diabetic male mice and age-matched controls were examined for measures of skeletal integrity. We assessed 1) the moment of inertia (I(MIN)) of the cortical bone within diaphysis, trabecular bone architecture of the metaphysis, and mineralization density of the tissue (TMD) for each compartment of the femur by micro-computed tomography and 2) biomechanical properties by three-point bending test (femur) and nanoindentation (tibia). In the metaphysis, a significant decrease in trabecular bone volume fraction and trabecular TMD was apparent after 10 weeks of diabetes. For cortical bone, type 1 diabetes was associated with decreased cortical TMD, I(MIN), rigidity, and peak moment as well as a lack of normal age-related increases in the biomechanical properties. However, there were only modest differences in material properties between diabetic and normal mice at both whole bone and tissue-levels. As the duration of diabetes increased, bone toughness decreased relative to control. If the sole effect of diabetes on bone strength was due to a reduction in bone size, then I(MIN) would be the only significant variable explaining the variance in the maximum moment. However, general linear modeling found that the relationship between peak moment and I(MIN) depended on whether the bone was from a diabetic mouse and the duration of diabetes. Thus, these findings suggest that the elevated fracture risk among diabetics is impacted by complex changes in tissue properties that ultimately reduce the fracture resistance of bone.