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In biomedical and preclinical research, the current standard method for measuring blood perfusion inside murine bone, radiolabeled microspheres, is a terminal procedure that cannot be used to monitor longitudinal perfusion changes. Laser Doppler flowmetry (LDF) can quantify perfusion within the proximal tibial metaphysis of mice in vivo but requires a surgical procedure to place the measurement probe directly onto the bone surface.Sustained inflammation for over a month following this technique was previously reported, and previous studies have used LDF as an endpoint-only procedure. We developed a modified, minimally invasive LDF procedure to measure intraosseous perfusion in the murine tibia without stimulating local or systemic inflammation or inducing gait abnormalities. This modified technique can be used to measure perfusion weekly for up to at least a month.• Unlike previous endpoint-only techniques, this modified LDF procedure can be performed weekly to monitor serial changes to intraosseous perfusion in the murine tibia • The modified LDF technique utilizes a smaller, more localized incision to minimize invasiveness and speed recovery Keywords bone blood flow, vascular supply 16 17 Modified LDF Procedure 18 This protocol was approved by the Institutional Animal Care and Use Committee at North 19 Carolina State University. In this study, the LDF procedure was performed on eighteen 20 14-week-old male C57Bl/6J mice (The Jackson Laboratory, Bar Harbor, ME). Mice should 21 be fasted 6-8 hours prior to the procedure. The LDF probe is sensitive to movement, 22 temperature, and light, so care should be taken to ensure consistent conditions across 23 9. Record perfusion with the LDF monitor's software. We capture 30-second 78 perfusion readings, which are sufficient for a stable average measurement, but 79 longer or shorter readings could be recorded, if desired. Note: Perfusion readings 80 under 5 PU indicate the probe is not placed perpendicular to the medullary cavity, 81 and readings over 30 perfusion units (PU) likely indicate the probe slipped off the 82 bone and is recording in soft tissue. A noisy signal (greater than 5 PU variation) 83 suggests that either the probe is not pressed firmly against the bone, the probe is 84 moving, or the probe is not perpendicular to a flat bone surface. In any of these 85 cases, reposition and secure the probe until resolved. Large spikes in the signal 86 likely indicate limb movement; sufficient anesthesia depth should be confirmed, 87 Figure 2: Image showing probe placement during the modified laser Doppler flowmetry technique on the right hindlimb. The arrow points to the approximate position of the right knee. micromanipulator LDF probe 5 mm and the hindlimb can be taped down above the knee if additional security is 88 needed. 89 10. After obtaining a good reading, remove and reposition the LDF probe, and record 90 again. Note: We recommend repositioning the probe at least one time to ensure 91 consistent placement over the bone. If the two readings are dissimilar...

The bottom‐up approach describes the synthesis of bulk materials from the finest possible length scales to obtain the best global properties. This approach was adapted to the synthesis of multi‐phase ceramic composites produced from metal oxides produced by liquid‐feed flame spray pyrolysis (LF‐FSP). The effect of length scale of mixing was tested through two processing schemes, mixed single metal‐oxide nanopowders (NPs) and nanocomposite NPs having the desired composition within single particles. For the Al2O3–Y2O3–ZrO2 ternary system, composites prepared from nanostructured nanoparticles sinter to finer grain sizes (<410 nm) at equivalent densities of 95%TD than those prepared from mixed nanoparticle processing. These contrast with our previous studies in this area where mixed NP processing gave the best or equivalent results. The nanocomposite NPs produced in this study exhibit novel nanostructures with three phases contained within single particles <26 nm average particle size (APS). This nanostructure may directly explain the enhanced sintering of the nanocomposite NPs and may provide an impetus for future synthesis of similarly structured NPs.

This effort contrasts "bottom-up" processing of YAG/a-Al 2 O 3 composites where both elements (as 40-50 nm APSs nanopowders) are present at close to atomic mixing with reactive sintering where ball-milled mixtures of the individual nanopowders (40-50 nm APSs) give uniform elemental mixing at length scales closer to 100-800 nm with correspondingly much longer diffusion distances. In contrast to expectations, densification with control of final grain sizes is best effected using reactive sintering. Thus, reactive sintering to densities ≥95% occurs at only 1500°C with final grain sizes of %1000 nm for all samples. In contrast "bottom up" processing to ≥95% densities is only achieved at 1600°C, and with final grain sizes of 1700 nm. The reason for this unexpected behavior is that YAG phase forms early in the bottom up approach greatly inhibiting diffusion promoted densification. In contrast, in reactive sintering, YAG is prevented from forming because of the longer diffusion distances such that densification occurs prior to full conversion of the Y 2 O 3 component to YAG. The found hardness values are statistically superior to literature values for composites near the known eutectic composition. In an accompanying paper, the addition of a third component reverses this behavior.

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