Rabbit cortical bone tissue increases its elastic stiffness but becomes less viscoelastic with age

Bone is dynamic tissue undergoing changes in its composition, structure and functional properties during growth. It has been proposed that especially changes in the collagen phase of bone are responsible for making the bone more fragile, and potentially less viscoelastic with age. Hence, robust methods to measure viscoelasticitiy are needed. This study aimed to characterize the development of the elastic and viscoelastic mechanical properties of rabbit bone during maturation and growth, as assessed by nanoindentation. The humeri from female New Zealand white rabbits of varying age (newborn, 11 days, 4 weeks, 3 and 6 months old, n=8 per group) were investigated. Mid-diaphyseal cortical bone samples were cut, dehydrated, embedded and polished. Nanoindentation probing, semi-dynamic testing with a frequency of 20 Hz and creep with a dwell time of 60 s were performed under load control to quantify the elastic and the time-dependent viscoelastic mechanical properties of bone. The elastic moduli were evaluated with all three methods and the viscoelastic parameters were assessed using the phase-shift and the creep time constant. The elastic stiffness of bone increased significantly with each consecutive age group, from 11 days to 6 months of age, based on the reduced modulus from the indentation probing, the storage modulus from the semi-dynamic test, and the first elastic parameter from the creep test. These elastic parameters correlated significantly (R=0.88-0.94, p<0.01). The values of viscoelastic parameters, the phase-shift and time creep constant, decreased significantly with age. The viscous properties determined by the creep and the semi-dynamic testing correlated significantly (R=0.90, p<0.01), however, no correlation was found between the phase-shift and the creep time constant. Additionally, the present results showed specific associations with tissue composition, as measured with Fourier Transform Infrared spectroscopy of the same samples. In summary, the present results reveal significant changes in material properties of rabbit cortical bone with age. The elastic modulus of bone tissue increased by approximately 60%, whereas the viscoelastic parameters decreased by 10% to 25% during the first 6 months of the rabbit's life. Together, this indicates significant structural and functional maturation of the bone matrix during growth of the rabbit.