4D Multimodality Imaging of <em>Citrobacter rodentium</em> Infections in Mice

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Caliper- a PerkinElmer Company

This study analyzes data from 206 CaP specimens (68 HA, 70 BCP, and 68 beta-TCP) fractured via biaxial flexure testing. Specimens were divided into four groups: (a) Group I, dry; (b) Group II, wet (day 0, immersion time approximately 5-10 s); (c) Group III, after immersion in media for 21 days (day 21); and (d) Group IV, after culturing osteoblasts (OBs) on the surface for 21 days (day 21 with cells). X-ray diffraction verified the presence of minor second phases in HA and beta-TCP while BCP was a biphasic mixture of HA and beta-TCP with minor phases present. The statistical significance (p < 0.05) of differences in the measured biaxial flexure fracture strength, S, between groups was assessed via one-way ANOVA with Tukey's test. Also, a two-parameter Weibull analysis assessed the mechanical reliability of each group. Osteoblasts increase the biaxial flexure fracture strength in a statistically significant way compared to both the HA discs in Groups II and III. Scanning electron microscope examination revealed grain boundary grooving on the sintered surfaces and with thermal expansion anisotropy, likely leads to the observed rapid strength decline upon exposure to media found in Groups II, III and IV.

Biaxial flexure testing of calcium phosphate bioceramics for use in tissue engineering.

PTH is in clinical use for the treatment of osteoporosis and is under intensive investigation for its potential in applications of tissue engineering, fracture healing, and implant integration. However, the mechanisms of its action to stimulate bone formation are still unclear. A novel bone tissue engineering model was used to elucidate basic mechanisms of PTH anabolic actions. Ectopic ossicles containing cortical bone, trabecular bone, and a hematopoietic marrow were generated from implanted bone marrow stromal cells (BMSC). One week after implantation, nude mice were administered PTH or vehicle for 1 week (group 1), 3 weeks (group 2), or 7 weeks (group 3). Another group was also treated for 3 weeks, initiated 12 weeks after implantation (group 4). Micro-radiography and histomorphometry revealed increased marrow cellularity in group 1 PTH-treated ossicles, increased bone in group 2 PTH-treated ossicles, and similar amounts of bone in both group 3 and 4 ossicles regardless of treatment. Incidence of phosphate mineral and phosphate mineral to hydroxyproline ratio via Raman spectroscopy were significantly higher after 3 weeks versus 1 week of PTH treatment, but there was no difference between PTH- and vehicle-treated ossicles. Early events of PTH action in group 1 ossicles and the effects of a single injection of PTH on 1- and 2-week-old ossicles were evaluated by Northern blot analysis. Osteocalcin (OC) mRNA was increased after 1 week of intermittent PTH treatment in ossicles and calvaria but an acute injection did not alter OC mRNA. In contrast, a single injection of PTH increased matrix gamma-carboxyglutamic acid protein (MGP) mRNA in 2-week-old ossicles. Differential and temporal-dependent effects of PTH on OC and MGP suggest at the molecular level, that PTH acts to inhibit osteoblast mineralization. However, this does not translate into tissue level alterations. These data indicate that anabolic actions of PTH in ectopic ossicles are temporally dependent on the BMSC implanted and suggest that cell implantation strategies are particularly responsive to PTH.

Anabolic actions of PTH (1-34): use of a novel tissue engineering model to investigate temporal effects on bone.

Thrombospondin 3 (TSP3) is structurally similar to cartilage oligomeric matrix protein (COMP/TSP5), but its function is unknown. To determine the functional significance of TSP3, we generated mice with a targeted disruption of Thbs3. TSP3-null mice are viable and fertile and show normal prenatal skeletal patterning, based on Alcian blue/Alizarin red S staining. However, subtle and transient abnormalities were detected in the developing postnatal skeleton. Young adult TSP3-null mice are heavier than controls, and analyses of the geometric and biomechanical properties of long bones show increases in the moments of inertia, endocortical and periostal radii, and failure load. The bones of 9-week-old TSP3-null male mice also have a significantly greater cortical area. Most of these differences were no longer detected in 15-week-old mice. Micro-computed tomography scans showed that the trabecular bone proximal to the femoral head growth plate developed at an earlier time in TSP3-null mice than in wild-type mice. Thus, vascular invasion and ossification start in the femoral heads of TSP3-null mice at 9 weeks, whereas the wild-type femoral head is still composed of hypertrophic chondroctyes in a calcified matrix at 15 weeks. These results provide evidence for a role for TSP3 in the regulation of skeletal maturation in mice.

Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head.

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs approximately 10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce-/- mice are viable and fertile, Pcolce-/- male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce-/- male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce-/- vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce-/- mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce-/- tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.

Procollagen C proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues.

PTH (1-34) is the only FDA-approved anabolic agent for osteoporosis treatment in the U.S., but its mechanisms are not completely understood. This study investigated PTH effects on osteogenic cells at various stages of differentiation and proliferation using an engineered bone growth model in vivo. Ossicles were generated from bone marrow stromal cells (BMSCs) implanted in immunocompromised mice. Three weeks of PTH (40 microg/kg/day) or vehicle treatment initiated 1 day, 1, 2, or 3 weeks after BMSC implantation resulted in an anabolic response in PTH-treated implants (via histomorphometry and muCT) in all treatment groups. A novel in vivo tracking strategy with luciferase tagged BMSCs and weekly bioluminescent imaging of ossicles revealed increased donor cell proliferation in PTH-treated ossicles. The greatest increase occurred during the first week, and the activity remained elevated in PTH-treated implants over time. Zoledronic acid (ZA) was combined with PTH to delineate interactive mechanisms of these bone active agents. Combining ZA with PTH treatment reduced the PTH-mediated increase in luciferase BMSC activity, serum osteocalcin, and serum tartrate resistant acid phosphotase-5b (TRAP-5b) but ZA did not reduce the PTH-induced increase in total bone. Since zoledronic acid reduced PTH-induced proliferation without reducing bone volume, these data suggest that combining PTH and bisphosphonate therapy warrants further investigation in the treatment of bone disorders.

Parathyroid hormone mediates bone growth through the regulation of osteoblast proliferation and differentiation.

Thrombospondin-2 (TSP2) is a matricellular protein with increased expression during growth and regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were examined 5, 10, and 20 days after fracture using microCT, histology, immunohistochemistry, quantitative RT-PCR, and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30% more bone by microCT and 40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor [HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture, chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important implications for expediting healing or promoting regeneration in nonunions.

Thrombospondin-2 influences the proportion of cartilage and bone during fracture healing.

Bone mineral density (BMD) measurements are critical in many research studies investigating skeletal integrity. For pre-clinical research, micro-computed tomography (microCT) has become an essential tool in these studies. However, the ability to measure the BMD directly from microCT images can be biased by artifacts, such as beam hardening, in the image. This three-part study was designed to understand how the image acquisition process can affect the resulting BMD measurements and to verify that the BMD measurements are accurate. In the first part of this study, the effect of beam hardening-induced cupping artifacts on BMD measurements was examined. In the second part of this study, the number of bones in the X-ray path and the sampling process during scanning was examined. In the third part of this study, microCT-based BMD measurements were compared with ash weights to verify the accuracy of the measurements. The results indicate that beam hardening artifacts of up to 32.6% can occur in sample sizes of interest in studies investigating mineralized tissue and affect mineral density measurements. Beam filtration can be used to minimize these artifacts. The results also indicate that, for murine femora, the scan setup can impact densitometry measurements for both cortical and trabecular bone and morphologic measurements of trabecular bone. Last, when a scan setup that minimized all of these artifacts was used, the microCT-based measurements correlated well with ash weight measurements (R(2)=0.983 when air was excluded), indicating that microCT can be an accurate tool for murine bone densitometry.

Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD.

Avances recientes en invasiva &oacute;pticos, radiogr&aacute;ficos y proyecci&oacute;n de imagen de &mu;CT proporcionan una oportunidad para supervisar procesos biol&oacute;gicos longitudinalmente en un contexto anat&oacute;mico. Una aplicaci&oacute;n particularmente relevante para combinar estas modalidades es el estudio de infecciones de implantes ortop&eacute;dicos. Estas infecciones se caracterizan por la formaci&oacute;n de biofilms bacterianos persistentes en los materiales implantados, causando inflamaci&oacute;n, osteolisis periprot&eacute;sica, osteomielitis y da&ntilde;os, en falta y aflojamiento del implante del hueso.

Monitoreo de carga bacteriana, inflamaci&oacute;n y hueso da&ntilde;an longitudinalmente con &oacute;ptica y proyecci&oacute;n de imagen de &mu;CT en una infecci&oacute;n del implante ortop&eacute;dico en ratones.

&#x6700;&#x65B0;&#x8FDB;&#x5C55;&#x975E;&#x4FB5;&#x5165;&#x5F0F;&#x5149;&#x5B66;&#x3001; &#x7167;&#x76F8;&#x548C; &mu;CT &#x6210;&#x50CF;&#x63D0;&#x4F9B;&#x673A;&#x4F1A;&#x76D1;&#x6D4B;&#x7EB5;&#x5411;&#x5728;&#x89E3;&#x5256;&#x5B66;&#x65B9;&#x9762;&#x7684;&#x751F;&#x7269;&#x8FC7;&#x7A0B;&#x3002;&#x7279;&#x522B;&#x662F;&#x6709;&#x5173;&#x7533;&#x8BF7;&#xFF0C;&#x7ED3;&#x5408;&#x8FD9;&#x4E9B;&#x65B9;&#x5F0F;&#x662F;&#x7814;&#x7A76;&#x9AA8;&#x79D1;&#x690D;&#x5165;&#x7269;&#x611F;&#x67D3;&#x3002;&#x8FD9;&#x4E9B;&#x611F;&#x67D3;&#x7684;&#x7279;&#x70B9;&#x662F;&#x5BF9;&#x690D;&#x5165;&#x6750;&#x6599;&#xFF0C;&#x9020;&#x6210;&#x708E;&#x75C7;&#x3001; &#x5047;&#x4F53;&#x5468;&#x56F4;&#x9AA8;&#x6EB6;&#x89E3;&#x3001; &#x9AA8;&#x9AD3;&#x708E;&#x3001; &#x6301;&#x4E45;&#x6027;&#x7EC6;&#x83CC;&#x751F;&#x7269;&#x819C;&#x7684;&#x5F62;&#x6210;&#x548C;&#x9AA8;&#x79CD;&#x690D;&#x4F53;&#x677E;&#x52A8;&#x548C;&#x6545;&#x969C;&#x6240;&#x9020;&#x6210;&#x7684;&#x635F;&#x5BB3;&#x3002;

&#x76D1;&#x6D4B;&#x7EC6;&#x83CC;&#x7684;&#x8D1F;&#x62C5;&#x3001; &#x708E;&#x75C7;&#x548C;&#x9AA8;&#x7834;&#x574F;&#x7EB5;&#x5411;&#x4F7F;&#x7528;&#x5149;&#x5B66;&#x548C; &mu;CT &#x6210;&#x50CF;&#x5728;&#x9AA8;&#x79D1;&#x690D;&#x5165;&#x7269;&#x611F;&#x67D3;&#x5C0F;&#x9F20;&#x3002;

Recent advances in non-invasive optical, radiographic and Î¼CT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.

Monitoring Bacterial Burden, Inflammation and Bone Damage Longitudinally Using Optical and Î¼CT Imaging in an Orthopaedic Implant Infection in Mice.

Recent advances in non invasif optiques, radiographiques et imagerie &mu;CT permettent de surveiller des processus biologiques longitudinalement dans un contexte anatomique. Une application particuli&egrave;rement pertinente pour la combinaison de ces modalit&eacute;s est d&#39;&eacute;tudier les infections implant orthop&eacute;dique. Ces infections sont caract&eacute;ris&eacute;es par la formation des biofilms bact&eacute;riens persistants sur les mat&eacute;riaux implant&eacute;s, provoquant une inflammation, ost&eacute;olyse p&eacute;riprosth&eacute;tique, ost&eacute;omy&eacute;lite et dommage, r&eacute;sultant en desserrant implant et l&#39;&eacute;chec d&#39;os.

Surveillance de la charge bact&eacute;rienne, l&#39;Inflammation et l&#39;OS endommagent longitudinalement l&#39;aide optique et imagerie &mu;CT dans une Infection de l&#39;Implant orthop&eacute;dique chez la souris.

J&uuml;ngste Fortschritte in der nicht-invasiven optisch, radiologischen und &Mu;ct Imaging bieten Gelegenheit, biologische Prozesse in L&auml;ngsrichtung in einem anatomischen Kontext zu &uuml;berwachen. Eine besonders relevante Anwendung f&uuml;r die Kombination dieser Modalit&auml;ten ist orthop&auml;dische Implantate Infektionen zu studieren. Diese Infektionen zeichnen sich durch die Bildung von persistente bakterielle Biofilme auf den implantierten Materialien, verursacht Entz&uuml;ndungen, Periprosthetic Osteolyse, Osteomyelitis, und Sch&auml;den im Implantat Lockerung und Ausfall die Knochendichte.

&Uuml;berwachung bakterielle Belastung, Schaden Entz&uuml;ndung und Knochen in L&auml;ngsrichtung mit optischen und &Mu;ct-Imaging in einer orthop&auml;dischen Implantat Infektion bei M&auml;usen.

I recenti progressi nella non-invasiva ottici, radiografici e &mu;CT imaging offrono la possibilit&agrave; di monitorare i processi biologici longitudinalmente in un contesto anatomico. Una applicazione particolarmente rilevante per la combinazione di queste modalit&agrave; &egrave; quello di studiare le infezioni di protesi ortopediche. Queste infezioni sono caratterizzate dalla formazione di biofilm batterici persistente sui materiali impiantati, provocando infiammazione, osteolisi PERIPROTESICHE, osteomielite e danni, con conseguente allentamento dell&#39;impianto e il fallimento dell&#39;osso.

Controllo carica batterica, infiammazione e danno osseo longitudinalmente utilizzando ottiche e &mu;CT Imaging in un ortopedico dell&#39;impianto infezione nei topi.

&#x5149;&#x5B66;&#x3001;x &#x7DDA;&#x64AE;&#x5F71;&#x306E;&#x6700;&#x8FD1;&#x306E;&#x975E;&#x4FB5;&#x8972;&#x7684;&#x9032;&#x6B69;&#x3068; &mu; CT &#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;, &#x7E26;&#x65AD;&#x7684;&#x306B;&#x89E3;&#x5256;&#x5B66;&#x7684;&#x6587;&#x8108;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x6A5F;&#x4F1A;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30E2;&#x30C0;&#x30EA;&#x30C6;&#x30A3;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x308B;&#x305F;&#x3081;&#x306B; 1 &#x3064;&#x306E;&#x7279;&#x306B;&#x95A2;&#x9023;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x3001;&#x6574;&#x5F62;&#x5916;&#x79D1;&#x30A4;&#x30F3;&#x30D7;&#x30E9;&#x30F3;&#x30C8;&#x611F;&#x67D3;&#x3092;&#x52C9;&#x5F37;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x611F;&#x67D3;&#x75C7;&#x708E;&#x75C7;, &#x30D7;&#x30ED;&#x30B9;&#x30C6;&#x30FC;&#x30B7;&#x30B9;&#x878D;&#x89E3;, &#x9AA8;&#x9AC4;&#x708E;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3057;&#x3066;&#x6CE8;&#x5165;&#x6750;&#x6599;&#x306B;&#x95A2;&#x3059;&#x308B;&#x6C38;&#x7D9A;&#x7684;&#x306A;&#x7D30;&#x83CC;&#x30D0;&#x30A4;&#x30AA; &#x30D5;&#x30A3;&#x30EB;&#x30E0;&#x306E;&#x5F62;&#x6210;&#x306B;&#x3088;&#x3063;&#x3066;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x308B;&#x3057;&#x3001;&#x9AA8;&#x306E;&#x640D;&#x50B7;&#x3001;&#x30A4;&#x30F3;&#x30D7;&#x30E9;&#x30F3;&#x30C8;&#x3092;&#x7DE9;&#x3081;&#x308B;&#x3068;&#x5931;&#x6557;&#x306E;&#x7D50;&#x679C;&#x3057;&#x307E;&#x3059;&#x3002;

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&#xAD11;&#xD559;, &#xBC29;&#xC0AC;&#xC120; &#xBE44; &#xCE68;&#xB7B5; &#xC801;&#xC758; &#xCD5C;&#xADFC; &#xBC1C;&#xC804; &#xBC0F; &mu;CT &#xC774;&#xBBF8;&#xC9D5; &#xD574;&#xBD80;&#xD559; &#xCEE8;&#xD14D;&#xC2A4;&#xD2B8;&#xC5D0;&#xC11C; longitudinally &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xAE30;&#xD68C;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xD615;&#xC2DD;&#xC740; &#xACB0;&#xD569; &#xD55C; &#xD2B9;&#xD788; &#xAD00;&#xB828; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xC815;&#xD615; &#xC678;&#xACFC; &#xC784; &#xD50C; &#xB780; &#xD2B8; &#xAC10;&#xC5FC;&#xC744; &#xC5F0;&#xAD6C; &#xD558;&#xB294;. &#xC774;&#xB7EC;&#xD55C; &#xAC10;&#xC5FC; &#xC77C;&#xC73C;&#xD0A4;&#xB294; &#xC5FC;&#xC99D;, periprosthetic osteolysis, osteomyelitis, &#xC774;&#xC2DD;&#xB41C; &#xC7AC;&#xB8CC;&#xC5D0; &#xC601;&#xAD6C;&#xC801;&#xC778; &#xC138;&#xADE0;&#xC131; biofilms&#xC758; &#xD615;&#xC131;&#xC5D0; &#xC758;&#xD574; &#xD2B9;&#xC9D5; &#xBC0F; &#xBF08; &#xC190;&#xC0C1;, &#xC784; &#xD50C; &#xB780; &#xD2B8; &#xC644;&#xD654; &#xBC0F; &#xC2E4;&#xD328;&#xC758; &#xACB0;&#xACFC;.

&#xC138;&#xADE0;&#xC131; &#xBD80;&#xB2F4; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;, &#xC5FC;&#xC99D;&#xACFC; &#xBF08; &#xC190;&#xC0C1; Longitudinally &#xAD11;&#xD559;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xACE0; &mu;CT &#xC774;&#xBBF8;&#xC9D5;&#xC5D0; &#xC950;&#xC5D0; &#xC788;&#xB294; &#xC815;&#xD615; &#xC678;&#xACFC; &#xC784; &#xD50C; &#xB780; &#xD2B8; &#xAC10;&#xC5FC;.

Jeffrey A Meganck

Preclinical Imaging

Caliper- a PerkinElmer Company

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Jeffrey A Meganck

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Preclinical Imaging

Caliper- a PerkinElmer Company

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Preclinical Imaging