Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing

Abstract

The role of osteoclast-mediated resorption during fracture healing was assessed. The impact of two osteoclast inhibitors with different mechanisms of action, alendronate (ALN) and denosumab (DMAB), were examined during fracture healing. Male human RANKL knock-in mice that express a chimeric (human/murine) form of RANKL received unilateral transverse femur fractures. Mice were treated biweekly with ALN 0.1 mg/kg, DMAB 10 mg/kg, or PBS (control) 0.1 ml until death at 21 and 42 days after fracture. Treatment efficacy assessed by serum levels of TRACP 5b showed almost a complete elimination of TRACP 5b levels in the DMAB-treated animals but only approximately 25% reduction of serum levels in the ALN-treated mice. Mechanical testing showed that fractured femurs from both ALN and DMAB groups had significantly increased mechanical properties at day 42 compared with controls. muCT analysis showed that callus tissues from DMAB-treated mice had significantly greater percent bone volume and BMD than did both control and ALN-treated tissues at both 21 and 42 days, whereas ALN-treated bones only had greater percent bone volume and BMC than control at 42 days. Qualitative histological analysis showed that the 21-and 42-day ALN and DMAB groups had greater amounts of unresorbed cartilage or mineralized cartilage matrix compared with the controls, whereas unresorbed cartilage could still be seen in the DMAB groups at 42 days after fracture. Although ALN and DMAB delayed the removal of cartilage and the remodeling of the fracture callus, this did not diminish the mechanical integrity of the healing fractures in mice receiving these treatments. In contrast, strength and stiffness were enhanced in these treatment groups compared with control bones.

Figure Figure 1

Osteoclast activity across the time course of fracture in responses to alendronate or denosumab treatments. (A) Measured systemic activities based on serum levels of averaged TRACP 5b for each drug group across the time of treatment. Error bars are depicted in the figure are for SD, and levels of significance between individual drug groups to the control are denoted by asterisks. Significance was between all groups at 6 wk of treatment and is denoted by the asterisk above the control. (B) Panoramic histological assessments of local osteoclast activity within callus tissues based on TRACP 5b staining within 21‐day callus tissues. Composite images of fracture calluses (magnification, ×40). Bottom panel shows osteoclasts morphology on bone/cartilage surface fro each of the treatment groups. (magnification, ×600). (C) Osteoclast counts per unit total callus area. Osteoclast counts were determined. Letters denote individual groups (C, control; D, denosumab) that show statistical significance to the group that is denoted.

Figure Figure 2

Callus structures at 21 and 42 days after fracture as assessed by μCT. (A) Cutaway views of representative μCT 3D reconstructions of fracture calluses at 21 and 42 days after fracture (coronal plane cutaway). (B) Representative μCT images of single transverse slices of fracture calluses at 21 and 42 days after fracture. Images of these individual slices were taken from the central region of each callus. Experimental groups and times after fracture are indicated in the figure.

Figure Figure 3

Graphical analysis of μCT measurements of fracture calluses. (A) Total bone volume (TV). (B) Total bone volume (BV). (C) Mean cross‐sectional area (CsAr). (D) Percent bone volume (BV/TV). (E) BMC based on calibration to a standard quantity of hydroxyapatite. For all graphs, error bars are ±SD. Cross indicates significance relative to the same treatment group at day 21. Dots indicate significance between ALN and DMAB treatment groups at the same time point. Asterisks indicate significance compared with the control at the same time point. Crosses indicate significance between time points for an individual experimental group. Significance is at p < 0.05.

Figure Figure 4

Results from ash analysis of fractured femurs after 21 and 42 days of treatment. (A) Ash analysis: left, dry weight; middle, ash weight; right, tissue mineralization as the ratio of ash weight to dry weight. *p < 0.05 vs. control. ^○ p < 0.05 vs. ALN. (B) Correlations between results from ash and μCT analyses in day 42 femurs. Femur ash weight was strongly correlated to callus vBMC as measured by μCT, both within and across all groups (r ² = 0.556).

Figure Figure 5

Graphical analysis of torsion test results. One set of comparisons measure the differences between the contralateral bones and fractured bones (A and C), thereby providing a direct comparison of the regain of biomechanical competence to its unfractured state. The second set of measurements is the comparisons between the different drug groups and control across the two times within the fractured bones (B and D). This provides a direct comparison of the varying efficiencies of the two treatments vs. the control on the mechanical properties within the fractured bones themselves. The measure of the overall mechanical strength of the healing bones is presented in A and B. (A) Comparison of torque to failure between fractured bones and intact contralateral bones. (B) Comparison of fractured bones at between times and between experimental groups. Crosses indicate significance relative to the same treatment group at day 21. Asterisks indicate significance relative to the control at the same time point. (C) Comparison of torsional rigidity between fractured bones and intact contralateral bones. (D) Comparison of torsional rigidity between times and between experimental groups. Crosses indicate significance relative to the same treatment group at day 21. Asterisks indicate significance relative to the control at the same time point. For all graphs, error bars are ±SD. All p < 0.5.

Figure Figure 6

Representative photographs showing postmechanical testing fracture location. (Left panel) Break through the callus typical of what is seen in the control group. (Right panel) Break outside the callus typical of the groups treated with DMAB.

Figure Figure 7

Representative histological samples of fracture calluses at days 21 and 42 after fracture (safranin‐O and fast green). (A) Representative micrographs from 21 days after fracture. (Top panel) Composite images of fracture calluses (magnification, ×40). (Middle panel) Magnification, ×100. (Bottom panel) Magnification, ×400. Boxed areas show areas that were selected for each set of progressive magnifications in the three panels. Single boxed area in the panel showing the ×400 micrograph from the DMAB‐treated group highlights an individual ring of osteoid within the empty lacunae where a hypertrophic chondrocyte had resided. Also of note are distinct areas of hypertrophic chondrocytes and mixed bone and cartilage trabeculae that are still present in ALN and DMAB bones. (B) Representative micrographs from 42 days after fracture. (Top panel) Composite images of fracture calluses (magnification, ×40). (Middle panel) Magnification, ×100. (Right panels) Micrographs from two separate DMAB‐treated specimens.