Metadi supreme

The tibias of 3-month-old mice from each group were dissected and fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer solution (pH 7.4) at room temperature. Four hours later, the samples were immersed in 0.1M cacodylate solution. The samples were then dehydrated in ascending concentrations of ethanol and embedded in methyl-methacrylate (MMA) resin. Microcloths with Metadi Supreme polycrystalline diamond suspensions of decreasing sizes (Buehler, Lake Bluff, IL) were used to polish the sample surfaces. The samples were coated with carbon for backscattered SEM analyses. For the resin-casted SEM, the samples were acid-etched with 37% phosphoric acid for 2–10 seconds and washed with 5.25% sodium hypochlorite for 5 minutes. The samples were then coated with gold and palladium for secondary electron image analyses. A JEOL JSM-6300 scanning electron microscope (JEOL Limited, Tokyo, Japan) was used to perform the analyses as reported previously [14 (link)].

A notched toughness test was performed on the right femora.^{(34 (link))} A sharp notch was cut through the anterior surface of the mid-diaphysis using a razor blade lubricated with 1μm diamond suspension (MetaDi Supreme, Buehler) and a custom-built jig. Razor cutting depth was controlled using a micrometer. Notched bones were tested to catastrophic failure in 3 point bending at 0.001mm/sec. Load and deflection were recorded during each test. Following fracture, bones were briefly soaked in diluted household bleach to remove marrow from the fracture surface and then dehydrated in a graded ethanol series. Fracture surfaces were imaged under back-scattering scanning electron microscopy (SEM, Hitachi S3200N) and analyzed using a custom MATLAB script to quantify bone geometry, half-notch and half-crack angles, and calculate maximum load (K_C.Max) and instability (K_C.Inst) toughness.^{(34 (link))} K_C.Max was calculated using the ultimate force and half-notch angle. K_C.Inst was calculated using the force at catastrophic fracture and instability half-crack angle. Notch and crack geometry met the assumptions of the thick-walled cylinder crack propagation model, with half notch/crack angles less than 110°.^{(34 (link))} Sample sizes were 7–10 per group.