Establishment of a Reliable Model to Study the Failure of Fracture Healing in Aged Mice

Abstract

The failure of fracture healing represents a substantial clinical problem. Moreover, aged patients demonstrate an elevated risk for failed bone healing. However, murine models to study the failure of fracture healing are established only in young adult animals. Therefore, the aim of this study was to develop a reliable model to study failed fracture healing in aged mice. After creation of a 1.8 mm segmental defect and periosteal resection, femora of aged mice (18-20 months) and young adult control mice (3-4 months) were stabilized by pin-clip fixation. Segmental defects were analyzed by means of biomechanics, X-ray and micro-computed tomography (µCT), as well as histomorphometric, immunohistochemical and Western blot analysis. After 10 weeks all animals showed a complete lack of osseous bridging, resulting in fracture healing failure. Segmental defects in aged mice revealed a reduced bone formation and vascularization when compared to young adult mice. This was associated with a decreased expression of bone formation markers. In addition, we detected a reduced number of tartrate-resistance acid phosphatase (TRAP)-positive osteoclasts and an elevated osteoprotegerin (OPG)/receptor activator of NF-ĸB ligand (RANKL)-ratio in aged animals, indicating a reduced osteoclast activity. Moreover, aged animals showed also an enhanced inflammatory response, characterized by an increased infiltration of macrophages within the callus tissue. Taken together, we herein report for the first time a reliable model to study fracture healing failure in aged mice. In the future, the use of this model enables to study novel therapeutic strategies and molecular mechanics of failed fracture healing during aging.