Differential Effects of Dabigatran and Warfarin on Bone Volume and Structure in Rats with Normal Renal Function

Abstract

Background: Warfarin, a widely used anticoagulant, is a vitamin K antagonist impairing the activity of vitamin K-dependent Bone Gla Protein (BGP or Osteocalcin) and Matrix Gla Protein (MGP). Because dabigatran, a new anticoagulant, has no effect on vitamin K metabolism, the aim of this study was to compare the impact of warfarin and dabigatran administration on bone structure and vascular calcification.

Methods: Rats with normal renal function received for 6 weeks warfarin, dabigatran or placebo. Bone was evaluated immuno-histochemically and hystomorphometrically after double labelling with declomycin and calcein. Aorta and iliac arteries were examined histologically.

Results: Histomorphometric analysis of femur and vertebrae showed significantly decreased bone volume and increased trabecular separation in rats treated with warfarin. Vertebra analysis showed that the trabecular number was higher in dabigatran treated rats. Osteoblast activity and resorption parameters were similar among groups, except for maximum erosion depth, which was higher in warfarin treated rats, suggesting a higher osteoclastic activity. Therefore, warfarin treatment was also associated with higher bone formation rate/bone surface and activation frequency. Warfarin treatment may cause an increased bone turnover characterized by increased remodelling cycles, with stronger osteoclast activity compared to the other groups. There were no differences among experimental groups in calcium deposition either in aortic or iliac arteries.

Conclusions: These findings suggest for the first time that dabigatran has a better bone safety profile than warfarin, as warfarin treatment affects bone by reducing trabecular size and structure, increasing turnover and reducing mineralization. These differences could potentially result in a lower incidence of fractures in dabigatran treated patients.

Fig 1. Regions of Interest (ROI) for histomorphometric analyses for femur (1A) and vertebra (1B).

For femur analysis, we analyzed the trabecular and cortical bone (in green) of the secondary spongiosa area between 2 and 4 mm distal to the growth plate-metaphyseal junction in the upper part of the diaphysis. Concerning the vertebrae, we selected lumbar vertebrae and considered the middle area in frontal sections to evaluate trabecular and cortical bone parameters.

Fig 2. Dabigatran preserves femur volume and structure.

Note the higher bone volume and trabecular thickness with lesser trabecular separation in rats treated with dabigatran compared to rats treated with warfarin. Bone of a representative normal control rat is also shown. (magnification 100X).

Fig 3. Significant histomorphometric results of structural and dynamic parameters in femur rats.

In warfarin treated rats we observed lower bone volume and trabecular thickness with increased trabecular separation compared to dabigatran treated and control rats. In addition, treatment with warfarin was associated with a significant increase of turnover parameters (i.e. BFR/BS) and, in particular, activation frequency was higher in warfarin treated rats versus dabigatran treated or control rats. Microarchitecture, cortical thickness and porosity were similar among groups. Only rats treated with warfarin showed an alteration of bone volume and structure, suggesting increased bone fragility compared to dabigatran and control rats pattern. For the meaning of abbreviations see Table 2. *p<0.05, **p< 0.01, ***p<0.001 vs control; # p<0.05, ##p< 0.01, ### p<0.001 vs dabigatran.

Fig 4. Significant histomorphometric results of cellular parameters in femur (upper panel) and vertebra (lower panel) analysis of rats.

The main alteration of cellular parameters in warfarin treated rats was the increased osteoclast activity (ES/BS), although in femur a decreased osteoblast activity (OS/BS) was also detected. ES/BS: Erosion Surface / Bone Surface; OS/BS: Osteoid Surface / Bone Surface. *p< 0.01 vs control; #p<0.001 vs dabigatran; ##p< 0.005 vs dabigatran.

Fig 5. Dabigatran preserves vertebral volume and structure.

According to what observed in the femur, analysis of vertebrae showed increased bone volume, trabecular thickness and number, with lesser trabecular separation in rats treated with dabigatran with respect to warfarin treated study animals. Bone of a representative normal control rat is also shown. No significant differences in microarchitecture were observed between study groups. Warfarin treated rats showed thinner trabeculae with increased separation in the marrow space, highlighting a potentially increased fragility, despite similar microarchitecture. (Magnification 100X).

Fig 6. Significant histomorphometric results of structural and dynamic parameters in vertebrae of rats.

Similar to the findings in femur, in warfarin treated rats we observed lower bone volume and trabecular thickness with increased trabecular separation compared to dabigatran treated and control rats. In addition, treatment with warfarin was associated with a significant increase of turnover parameters, i.e. BFR/BS and activation frequency. Microarchitecture, cortical thickness and porosity were similar among groups. Only rats treated with warfarin showed an alteration of bone volume and structure, suggesting increased bone fragility compared to dabigatran and control rats pattern. *p<0.05,** p< 0.01, ***p<0.001 vs control; #p<0.05, ## p< 0.01, ###p<0.001 vs dabigatran.