Microview

Day 4 (n=3) nanoparticle injected embryos were taken from the portable Styrofoam incubator and placed in a custom built polycarbonate imaging chamber. All imaging was completed on a GE Healthcare eXplore CT 120 scanner. Embryos were scanned at 50μm for 5 minutes with a total of 800 projections with a voltage of 80kV. Post-processing was completed in MicroView (GE Healthcare) for quantification of contrast levels and OsiriX (Apple) for three dimensional reconstructions. Contrast intensity gray scale values were converted to Hounsfield units (HU), correlating to bone material density (Badea, M, Holdsworth, Johnson, & Angiography, 2008 (link)) from calibration to a bone standard phantom with known gross densities reflective of cortical bone (SB3, GE). Contrast enhancement measurements reported reflect the attenuation within the embryo above that of the surrounding yolk and albumen attenuation.

Fixed femurs were scanned using a GE Explore Locus microcomputed tomography (μCT) system at a voxel resolution of 20 μm obtained from 720 views. Beam angle of increment was 0.5, and beam strength was set at 80 peak kV and 450 uA. Each run consisted of control (non-Ovx) and Ovx and Ovx + L. reuteri treated mouse bones, and a calibration phantom to standardize grayscale values and maintain consistency. On the basis of autothreshold and isosurface analyses of multiple bone samples, a fixed threshold (760) was used to separate bone from bone marrow. Bone measurements were blinded; thus, knowledge of what mouse condition the analyzed bone was from was unknown until the final data was pooled. Trabecular bone analyses were performed in a region of trabecular bone defined at 1% of the total length (~0.17 mm) proximal to the growth plate of and extending 2 mm toward the diaphysis excluding the outer cortical bone. Trabecular bone mineral content, bone volume fraction, thickness, spacing, and number values were computed by a GE Healthcare MicroView software application for visualization and analysis of volumetric image data. Cortical measurements were performed in a 2 X 2 X 2 mm cube centered midway down the length of the bone using a threshold of 1000 to separate bone from marrow.

Tissue mineral density (TMD) was measured with microCT at a 50 micron voxel size (eXplore CT 120, GE Healthcare, Waukesha, WI, USA). A mineral phantom was used for calibration with analysis completed in Microview (version ABA 2.2, GE Healthcare, Waukesha, WI, USA).