Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

Vibration of osteoblastic cells using a novel motion-control platform does not acutely alter cytosolic calcium, but desensitizes subsequent responses to extracellular ATP.

Low-magnitude high-frequency mechanical vibration induces biological responses in many tissues. Like many cell types, osteoblasts respond rapidly to certain forms of mechanostimulation, such as fluid shear, with transient elevation in the concentration of cytosolic free calcium ([Ca2+ ]i ). However, it is not known whether vibration of osteoblastic cells also induces acute elevation in [Ca2+ ]i . To address this question, we built a platform for vibrating live cells that is compatible with microscopy and microspectrofluorometry, enabling us to observe immediate responses of cells to low-magnitude high-frequency vibrations. The horizontal vibration system was mounted on an inverted microscope, and its mechanical performance was evaluated using optical tracking and accelerometry. The platform was driven by a sinusoidal signal at 20-500 Hz, producing peak accelerations from 0.1 to 1 g. Accelerometer-derived displacements matched those observed optically within 10%. We then used this system to investigate the effect of acceleration on [Ca2+ ]i in rodent osteoblastic cells. Cells were loaded with fura-2, and [Ca2+ ]i was monitored using microspectrofluorometry and fluorescence ratio imaging. No acute changes in [Ca2+ ]i or cell morphology were detected in response to vibration over the range of frequencies and accelerations studied. However, vibration did attenuate Ca2+ transients generated subsequently by extracellular ATP, which activates P2 purinoceptors and has been implicated in mechanical signaling in bone. In summary, we developed and validated a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Vibration did not elicit acute elevation of [Ca2+ ]i , but did desensitize responses to later stimulation with ATP.

Practical fabrication of microfluidic platforms for live-cell microscopy.

We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.

Examining Practitioners' Assessments of Perceived Aesthetic and Diagnostic Quality of High kVp-Low mAs Pelvis, Chest, Skull, and Hand Phantom Radiographs.

This study investigated the usefulness of the dose optimization strategy of increased tube voltage (kVp) and decreased tube current-exposure time product (mAs) (or high kVp-low mAs) by examining practitioners' assessments of perceived aesthetic and diagnostic quality of direct digital radiographs acquired using this strategy. Ninety-one practitioners (radiologists, radiology residents, radiographers, and radiography students) from eight clinical sites in Ontario examined three types of radiographs ("standard" image, +20 kVp image, and +30 kVp image) for anthropomorphic pelvis, chest, skull, and hand phantoms and rated (on a five-point scale) each image in regard to its perceived aesthetic quality, perceived diagnostic quality, and visualization of anatomic structures. Our primary findings are that for the pelvis, skull, and hand-although not the chest-images acquired using standard technical factors were rated significantly higher in diagnostic and aesthetic quality than those acquired using the high kVp-low mAs strategy. Despite this, both standard and dose-optimized images of the pelvis, skull, and hand were rated to be of acceptable diagnostic quality for clinical use. In conclusion, for the pelvis, skull, and hand, an increase of 20 kVp was an effective strategy to reduce dose while still acquiring images of diagnostic quality.

Regional measurements of surface deviation volume in worn polyethylene joint replacement components.

Total joint replacements can be subject to the loss of polyethylene material due to wear, leading to osteolysis and decreased implant longevity. Micro-computed tomography (micro-CT) techniques have recently been developed to calculate 3D surface deviations in worn implant components. We describe a micro-CT technique to measure the volume of the surface deviations (volume of wear plus creep) within a specific region or compartment, and report its repeatability and reproducibility. Six worn polyethylene tibial inserts were scanned using a laboratory micro-CT scanner and subsequently reconstructed at 50 µm voxel spacing. A previously developed custom software application was used to quantify the 3D surface deviations between the worn tibial inserts and an unworn reference geometry. Three observers (two trained and one expert) used new custom software to manually outline the localized regions of surface deviation (three times for each of the worn inserts) and calculate the volume of the deviations. The overall intraobserver variability in the surface deviation volumes was 3.6% medially and 1.1% laterally. The overall interobserver variability was 4.8% medially and 1.7% laterally. Placement of points in outlining the region of deviation contributed the greatest variability to the measurements. Repeatability and reproducibility of the volume measurements are similar to measurements of total (nonregional) wear volume including a previous micro-CT technique (10%), fluid displacement (4.8%), and radiographic measurements (15.7%). The principles of this technique can likely be used to measure regional wear and creep volume in knee and hip joint replacement components from wear simulator, pin-on-disk, and retrieval studies.