Does Anticoagulant Medication Alter Fracture-Healing? A Morphological and Biomechanical Evaluation of the Possible Effects of Rivaroxaban and Enoxaparin Using a Rat Closed Fracture Model

Abstract

Low molecular weight heparin (LMWH) is routinely used to prevent thromboembolism in orthopaedic surgery, especially in the treatment of fractures or after joint-replacement. Impairment of fracture-healing due to increased bone-desorption, delayed remodelling and lower calcification caused by direct osteoclast stimulation is a well-known side effect of unfractioned heparin. However, the effect of LMWH is unclear and controversial. Recent studies strongly suggest impairment of bone-healing in-vitro and in animal models, characterized by a significant decrease in volume and quality of new-formed callus. Since October 2008, Rivaroxaban (Xarelto) is available for prophylactic use in elective knee- and hip-arthroplasty. Recently, some evidence has been found indicating an in vitro dose independent reduction of osteoblast function after Rivaroxaban treatment. In this study, the possible influence of Rivaroxaban and Enoxaparin on bone-healing in vivo was studied using a standardized, closed rodent fracture-model. 70 male Wistar-rats were randomized to Rivaroxaban, Enoxaparin or control groups. After pinning the right femur, a closed, transverse fracture was produced. 21 days later, the animals were sacrificed and both femora harvested. Analysis was done by biomechanical testing (three-point bending) and micro CT. Both investigated substances showed histomorphometric alterations of the newly formed callus assessed by micro CT analysis. In detail the bone (callus) volume was enhanced (sign. for Rivaroxaban) and the density reduced. The bone mineral content was enhanced accordingly (sign. for Rivaroxaban). Trabecular thickness was reduced (sign. for Rivaroxaban). Furthermore, both drugs showed significant enlarged bone (callus) surface and degree of anisotropy. In contrast, the biomechanical properties of the treated bones were equal to controls. To summarize, the morphological alterations of the fracture-callus did not result in functionally relevant deficits.

Fig 1. Experimental Setup.

(a) Standardized fracture: Anesthetized animal placed on the fracture device. Leg placed across an open platform. (b) Postoperative radiograph, a.p. view after fracture. A transverse fracture with minimal dislocation can be seen at the middle of the femoral shaft. (c) Setting during biomechanical testing of the specimens. The distance between the bars was adapted for each bone. All bones were loaded until failure (V-max) with a persistent test velocity of 5mm/min. Meanwhile a load-displacement diagram was recorded every 0.1 second and thereby failure load was determined. (d) Scout view scan before μCT-measurement.

Fig 2. Biomechanical Parameters.

(a, b) Load-displacement diagram of corresponding bones during three-point bending. The first diagram (a) shows the fracture-curve of the control side, the second the experimental side (b). The ordinate displays the force (N), the abscisse the displacement (in mm). Different scales of the ordinate. The red line in Fig 2b displays stiffness (gradient of the linear part of the load-displacement curve). The light blue area is the work to failure (area under the curve (Nmm)).

Fig 3. Results Biomechanical Testing, V-max absolute and ratio.

(a) Dot-plots of absolute V-max values for controls, Rivaroxaban and Enoxaparin; Control sides (unfractured femur) and experimental sides (fracture). No sign. differences between controls and substances. (b) Dot-plot of ratios fractured to unfractured bones in V-max for controls, Rivaroxaban and Enoxaparin. No sign. differences between controls and substances.

Fig 4. Results Biomechanical Testing, work to failure.

Dot-plots of work to failure (Nmm) for controls, Rivaroxaban and Enoxaparin; experimental sides (fracture). No sign. differences between controls and substances.

Fig 5. Micro-CT Scan, 3D reconstruction within the ROI.

μCT scans, 3-dimensional reconstruction of ROI, virtually sliced in half or axial cuts. (a, b) representative specimen of the control group. Lower callus volume in comparison to Rivaroxaban/Enoxaparin (representative specimen, here Rivaroxaban, pictures (c) and (d)).

Fig 6. Summary Micro-CT Scan.

Micro-CT based assessment of histomorphometry, black points indicate exact data, grey arbour mean data ± standard deviation. (a) Bone Volume BV (mm³) = mineralized callus volume: Significant difference of Rivaroxaban compared to control group (p = 0,004). (b) Bone Mineral Content = BMC defined as callus BV multiplied by TMD (mg hydroxyapatite/ ccm): Significant difference of Rivaroxaban compared to control group (p < 0,05). (c) Degree of Anisotropy = DA: Significant difference of both substances compared to control group (p < 0,05). (d) Trabecular Thickness = Tb-Th (mm): Significant difference of Rivaroxaban compared to control group (p < 0,05). (e) Bone Surface = BS (mm²): Significant difference of both substances compared to control group (p < 0, 05).