The effects of glucocorticoid on microarchitecture, collagen, mineral and mechanical properties of sheep femur cortical bone
– Validation of large animal model for tissue engineering and biomaterial research

Ming Ding,1* Carl Christian Danielsen,2 Søren Overgaard1

1Orthopaedic Research Laboratory, Department of Orthopaedics and Traumatology, Odense University Hospital, Institute of Clinical Research, University of Southern Denmark, Odense C, Denmark
2Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark

Osteopenia in sheep has been successfully induced by glucocorticoid treatment and the changes in properties of cancellous bone were comparable with those observed in humans after long-term glucocorticoid treatment. However, the influence on cortical bone has not been thoroughly elucidated.
This study aimed to investigate the influence of glucocorticoid on sheep cortical bone after long-term treatment. Specifically, we quantify the microarchitecture, mechanical properties, collagen and mineral quality of sheep cortical bone. We hypothesized that glucocorticoid treatment also had significant influences on cortical bone that might increase risk of fracture.
In this study, 18 female skeletal mature sheep were randomly allocated into 3 groups of 6 each. Group 1 (glucocorticoid-1) received prednisolone treatment (0.60 mg/kg/day, 5 times weekly) for 7 months. Group 2 (glucocorticoid-2) received the same treatment regime followed by observation of 3 months without treatment. Group 3 was left untreated and served as the controls. All sheep received restricted diet with low calcium and phosphorus. At sacrifice, cortical bone samples from the femur midshaft of sheep were harvested, micro-CT scanned and tested in 3 point bending and in tensile. Bone collagen and mineral were determined.
Cortical porosity was significantly increased in the glucocorticoid-2 compared with the glucocorticoid-1 and the controls. Apparent density was significantly decreased in the glucocorticoid-2 compared with the glucocorticoid-1. Collagen content was significantly increased in the glucocorticoid-2 compared with the glucocorticoid-1 and the controls. Bone mineral content did not differ between the groups.
Neither the three-point bending mechanical properties, nor the tensile mechanical properties differed significantly between the groups, while there was a trend towards decreasing bending mechanical properties in the glucocorticoid-2.
In conclusion, 7 months glucocorticoid treatment with malnutrition had significant impact on cortical microarchitecture of sheep femur midshaft. These changes occurred particularly 3 months after the glucocorticoid cessation suggesting a delayed effect of glucocorticoid on cortical bone. Thus, changes in cortical bone beyond cancellous bone might further increase fracture risk.

Key works: glucocorticoid induced osteoporosis; microarchitecture; mechanical property; collagen and mineral; sheep cortical bone; tissue engineering