Evaluation of fetal bone structure and mineralization in IGF-I deficient mice using synchrotron radiation microtomography and Fourier transform infrared spectroscopy.

ABSTRACT

The role of insulin like growth factor-I (IGF-I) during pre-natal development has not been evaluated in detail. However, the high degree of growth retardation and peri-natal mortality in IGF-I deficient mouse models indicates that it plays a critical role during this time. Techniques to assess the structure and quality of bone in small animal fetuses could be beneficial in better understanding its role in bone metabolism and skeletal development. Synchrotron microtomography (SR-microCT) and Fourier transform infrared spectroscopy (FTIR) may provide methods to visualize and quantify differences in the structure and mineral density of bone in small animal fetuses. Tibia and spine from IGF-I deficient and wildtype fetal mice (18th day gestation) were imaged using SR-microCT. Three-dimensional structural indices and the degree of mineralization were determined for each sample. Mineralization was also assessed using FTIR and von Kossa staining. Bone volume was systematically lower in IGF-I -/- animals (tibia -15%, p<0.05) while both sites were found to have a more rod-like architecture (24%, p<0.05; 113%, p<0.01) and lower trabecular separation (-16%, p<0.05; -21%, p<0.05). These structural results were mostly consistent with those seen in adult models of IGF-I deficiency. The degree of mineralization as measured by SR-microCT was higher in the IGF-I tibial metaphysis (11.7%, p<0.0001), while FTIR of the whole bone showed mineralization to be lower in the knockout group (-11%, p<0.05). Interestingly, von Kossa staining revealed no mineral content in the IGF-I -/- spinal ossification center while SR-microCT clearly indicated the presence of highly attenuating components, if somewhat lower in IGF-I -/- animals (-2.2%, p<0.05). This indicates that IGF-I deficiency is linked to subtle differences in the mineral environment and mineralization progression. The advantages unique to SR-microCT allow for 3D visualization and quantification of pre-natal bone microstructure and mineral density in mice which was not previously possible.