RAPAMYCIN AFFECTS EARLY FRACTURE HEALING IN MICE BY INHIBITING VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) EXPRESSION DURING ENDOCHONDRAL OSSIFICATION

Abstract

The immunosuppressive drug rapamycin (RAPA) prevents rejection in organ transplantation by inhibiting interleukin-2-stimulated T-cell division. RAPA has also been suggested to possess strong anti-angiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF). Because VEGF is a key growth factor in fracture healing, the present study was conducted to analyze the effect of RAPA on bone repair.

For the herein introduced study 35 SKH-1Hr mice were treated by a daily intraperitoneal (i.p.) injection of RAPA (1.5mg/kg/d) from the day of fracture until sacrifice. Two or five weeks after fracture, animals were killed and bone healing was analyzed using radiological (n=16 at 2 weeks; n=16 at 5 weeks), biomechanical (n=2x8), and histomorphometric (n=2x8)

Methods: At 2 weeks additional animals were studied to achieve tissue for protein biochemical analysis of VEGF and proliferating cell nuclear antigen (PCNA; n=3). Additional 34 mice, which received the vehicle only, served as controls. Analyses in controls were similar to those of RAPA-treated animals.

X-ray analyses demonstrated that RAPA treatment inhibits callus formation after 2 weeks of fracture healing. The radiologically observed lack of callus formation after RAPA treatment was confirmed by histomorphometric analyses, which revealed a significantly diminished callus size and a reduced amount of bone formation when compared to vehicle-treated controls. Biomechanical testing further demonstrated that RAPA significantly reduces torsional stiffness of the callus (11.5±5.9% of the contralateral unfractured femur vs. 28.3±13.9% in controls; p< 0.05). Of interest, this was associated with a decrease of callus VEGF and PCNA expression. After 5 weeks of fracture healing, however, the negative impact of RAPA on fracture healing was found blunted and the radiological, histomorphometric and biomechanical differences observed after 2 weeks could not longer be detected.

We demonstrate that RAPA treatment leads to a severe alteration of early fracture healing. The negative action of RAPA on fracture repair at 2 weeks is most probably due to an inhibition of VEGF expression within the callus as suggested by the results of the Western blot analysis, demonstrating during the early phase of fracture healing a significantly reduced expression of VEGF and PCNA after RAPA treatment. This indicates a substantial alteration of cell proliferation and angiogenic vascularization during initial fracture healing. Since T-cells contribute to delayed fracture healing, RAPA may promote bone healing at later stages due to a reduction of interleukin-2-stimulated Tcell division.