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Diffuse idiopathic skeletal hyperostosis (DISH) is a prevalent noninflammatory spondyloarthropathy characterized by ectopic mineral formation along the anterolateral aspect of the vertebral column, yet little is known about its underlying pathogenesis. Our objective was to evaluate the histopathological features and composition of ectopic mineral within spinal tissues affected by DISH in humans. Thoracic spine segments from six embalmed cadaveric donors (one female and five males; median age 82 years) meeting the radiographic diagnostic criteria for DISH were evaluated using radiological, histological, and physical analyses. Overall, the histological features of ectopic mineralization at individual motion segments were heterogeneous, including regions of heterotopic ossification and dystrophic calcification. Heterotopic ossifications were characterized by woven and lamellar bone, multifocal areas of metaplastic cartilage, and bony bridges along the anterior aspect of the intervertebral disc space. Dystrophic calcifications were characterized by an amorphous appearance, a high content of calcium and phosphorus, an X-ray diffraction pattern matching that of hydroxyapatite, and radiodensities exceeding that of cortical bone. Dystrophic calcifications were found within the anterior longitudinal ligament and annulus fibrosus in motion segments both meeting and not meeting the radiographic criteria for DISH. In summary, our findings indicate that in DISH, ectopic mineral forms along the anterior aspect of the spine by both heterotopic ossification and dystrophic calcification of fibrocartilaginous tissues. Although both types of ectopic mineralization are captured by current radiographic criteria for DISH, dystrophic calcification may reflect a distinct disease process or an early stage in the pathogenesis of DISH.

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1−/−) exhibit progressive ectopic calcification of spinal tissues—a phenotype resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Our objective was to investigate potential calcification of orofacial tissues in ENT1−/− mice. Heads of wild-type mice and ENT1−/− mice from 3 to 17 months were evaluated using microcomputed tomography (μCT). Some heads were decalcified and processed for histological assessment. Other heads were examined using energy dispersive X-ray spectroscopy and micro X-ray diffraction. Using μCT, ENT1−/− mice showed extensive radiopaque lesions within the mandibular symphysis, the severity of which increased with advancing age. Histologically, at 6 months these ectopic radiopacities were found to correspond to acellular, amorphous, eosinophilic material, with no evidence of inflammatory cells. Because lesions were localised to the symphysis, we identified early pathological changes at 3 months and observed that lesions initiated specifically within the fibrocartilage pad. Energy-dispersive X-ray spectroscopy of ectopic lesions revealed large amounts of calcium and phosphorous in a molar ratio of ~1.59, and X-ray diffraction profiles matched that of calcium-deficient hydroxyapatite. This is the first characterisation of ectopic calcifications within the mandibular symphysis of ENT1−/− mice, indicating a role for ENT1 and adenosine metabolism in regulating calcification of fibrocartilaginous tissues. Moreover, these murine lesions resemble areas of dystrophic calcification in the spinal tissues of humans with DISH. Importantly, ectopic calcifications develop in a reproducible temporal pattern within a well-defined anatomical region and, thus, provide a model for determining the cellular and molecular pathways underlying ectopic calcification in DISH and related disorders.

Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues during development, physiological remodeling, and pathological bone loss. Osteoclasts have the ability to resorb substrate while concurrently migrating. However, the subcellular processes underlying migration are not well understood. It has been proposed that, in other cell types, cytosolic free Ca2+ concentration ([Ca2+]i) regulates cell protrusion as well as retraction. Integration of these distinct events would require precise spatiotemporal patterning of subcellular Ca2+. The large size of osteoclasts offers a unique opportunity to monitor patterns of Ca2+ during cell migration. We used ratiometric imaging to map [Ca2+]i within rat and mouse osteoclasts. Migration was characterized by lamellipodial outgrowth at the leading edge, along with intermittent retraction of the uropod. Migrating osteoclasts displayed elevation of [Ca2+]i in the uropod, that began prior to retraction. Dissipation of this [Ca2+]i gradient by loading osteoclasts with the Ca2+ chelator BAPTA abolished uropod retraction, on both glass and mineralized substrates. In contrast, elevation of [Ca2+]i using ionomycin initiated prompt uropod retraction. To investigate downstream effectors, we treated cells with calpain inhibitor-1, which impaired uropod retraction. In contrast, lamellipodial outgrowth at the leading edge of osteoclasts was unaffected by any of these interventions, indicating that the signals regulating outgrowth are distinct from those triggering retraction. The large size of mature, multinucleated osteoclasts allowed us to discern a novel spatiotemporal pattern of Ca2+ involved in cell migration. Whereas localized elevation of Ca2+ is necessary for uropod retraction, lamellipod outgrowth is independent of Ca2+—a heretofore unrecognized degree of specificity underlying the regulation of osteoclast migration. © 2014 American Society for Bone and Mineral Research.

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types. We have shown previously that disruption of P2X7 receptor function results in downregulation of osteogenic markers and upregulation of adipogenic markers in calvarial cell cultures. In the present study, we assessed whether loss of P2X7 receptor function results in changes to adipocyte distribution and lipid accumulation in vivo. Male P2X7 loss-of-function (KO) mice exhibited significantly greater body weight and epididymal fat pad mass than wild-type (WT) mice at 9 months of age. Fat pad adipocytes did not differ in size, consistent with adipocyte hyperplasia rather than hypertrophy. Histological examination revealed ectopic lipid accumulation in the form of adipocytes and/or lipid droplets in several non-adipose tissues of older male KO mice (9–12 months of age). Ectopic lipid was observed in kidney, extraorbital lacrimal gland and pancreas, but not in liver, heart or skeletal muscle. Specifically, lacrimal gland and pancreas from 12-month-old male KO mice had greater numbers of adipocytes in perivascular, periductal and acinar regions. As well, lipid droplets accumulated in the renal tubular epithelium and lacrimal acinar cells. Blood plasma analyses revealed diminished total cholesterol levels in 9- and 12-month-old male KO mice compared with WT controls. Interestingly, no differences were observed in female mice. Moreover, there were no significant differences in food consumption between male KO and WT mice. Taken together, these data establish novel in vivo roles for the P2X7 receptor in regulating adipogenesis and lipid metabolism in an age- and sex-dependent manner.

Synthetic-aperture radar (SAR) can be used to remotely estimate ground target vibrations by exploiting the Doppler in the returned signals. Recent studies suggest that time-frequency signal-processing tools can retrieve the vibration signature from the returned SAR signals. A vibration estimation method based on the fractional Fourier transform (FRFT) was reported earlier and it was tested on simulated SAR data. In this paper, a first-time demonstration of the FRFT-based vibration estimation method is reported using real SAR data collected by the Lynx (K u -band) SAR system. The vibrating target is an aluminum triangular trihedral with lateral length of 15 inches. The FRFT-based algorithm is shown to successfully retrieve a 3 mm peak-to-peak amplitude, 5 Hz vibration of the target from real SAR data.