Biomechanical evaluation of combined short segment fixation and augmentation of incomplete osteoporotic burst fractures.

BACKGROUND: Treating traumatic fractures in osteoporosis is challenging. Multiple clinical treatment options are found in literature. Augmentation techniques are promising to reduce treatment-related morbidity. In recent years, there have been an increasing number of reports about extended indication for augmentation techniques. However, biomechanical evaluations of these techniques are limited. METHODS: Nine thoracolumbar osteoporotic spinal samples (4 FSU) were harvested from postmortem donors and immediately frozen. Biomechanical testing was performed by a robotic-based spine tester. Standardized incomplete burst fractures were created by a combination of osteotomy-like weakening and high velocity compression using a hydraulic material testing apparatus. Biomechanical measurements were performed on specimens in the following conditions: 1) intact, 2) fractured, 3) bisegmental instrumented, 4) bisegmental instrumented with vertebroplasty (hybrid augmentation, HA) and 5) stand-alone vertebroplasty (VP). The range of motion (RoM), neutral zone (NZ), elastic zone (EZ) and stiffness parameters were determined. Statistical evaluation was performed using Wilcoxon signed-rank test for paired samples (p = 0.05). RESULTS: Significant increases in RoM and in the NZ and EZ (p < 0.005) were observed after fracture production. The RoM was decreased significantly by applying the dorsal bisegmental instrumentation to the fractured specimens (p < 0.005). VP reduced fractured RoM in flexion but was still increased significantly (p < 0.05) above intact kinematic values. NZ stiffness (p < 0.05) and EZ stiffness (p < 0.01) was increased by VP but remained lower than prefracture values. The combination of short segment instrumentation and vertebroplasty (HA) showed no significant changes in RoM and stiffness in NZ in comparison to the instrumented group, except for significant increase of EZ stiffness in flexion (p < 0.05). CONCLUSIONS: Stand-alone vertebroplasty (VP) showed some degree of support of the anterior column but was accompanied by persistent traumatic instability. Therefore, we would advocate against using VP as a stand-alone procedure in traumatic fractures. HA did not increase primary stability of short segment instrumentation. Some additional support of anterior column and changes of kinematic values of the EZ may lead one to suppose that additive augmentation may reduce the load of dorsal implants and possibly reduce the risk of implant failure.

Experimentally induced incomplete burst fractures - a novel technique for calf and human specimens.

BACKGROUND: Fracture morphology is crucial for the clinical decision-making process preceding spinal fracture treatment. The presented experimental approach was designed in order to ensure reproducibility of induced fracture morphology. RESULTS: The presented method resulted in fracture morphology, found in clinical classification systems like the Magerl classification. In the calf spine samples, 70% displayed incomplete burst fractures corresponding to type A3.1 and A3.2 fractures. In all human samples, superior incomplete burst fractures (Magerl A3.1) were identified by an independent radiologist and spine surgeon. CONCLUSIONS: The presented set up enables the first experimental means to reliably model and study distinct incomplete burst fracture patterns in an in vitro setting. Thus, we envisage this protocol to facilitate further studies on spine fracture treatment of incomplete burst fractures.

Differentiation of angiogenic burden in human cancer xenografts using a perfusion-type optical contrast agent (SIDAG).

INTRODUCTION: Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium). METHOD: Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined. RESULTS: SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 +/- 90 nmol/l versus 49 +/- 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 +/- 0.9% versus 0.8 +/- 0.53%; P < 0.01), blood vessel profile density 5-fold (399 +/- 36 BVPs/mm2 versus 78 +/- 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours. CONCLUSION: Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.

Prediction of antiangiogenic treatment efficacy by iron oxide enhanced parametric magnetic resonance imaging.

RATIONALE AND OBJECTIVES: Tools for monitoring modern target-specific antiangiogenic and antivascular therapies are highly desirable because treatment strategies are time consuming, expensive, and yet sometimes ineffective. Therefore, the aim of this experimental study was to evaluate the predictive value of steady-state ultrasmall particles of iron oxide (USPIO; SH U 555 C)-enhanced magnetic resonance imaging (MRI) for early assessment of antivascular tumor-treatment effectiveness. METHODS: Mice were inoculated with an HT-1080 fibrosarcoma xenograft and subjected to target-specific antivascular therapy using a selective thrombogenic vascular-targeting agent (truncated tissue factor fused to RGD peptide) or saline as control. Four to 8 hours after treatment, the USPIO-induced change in the transverse relaxation rate DeltaR2* was measured by MRI, and the vascular volume fraction (VVF) was calculated by calibrating DeltaR2* of the tumor by DeltaR2* of muscle tissue. Treatment response was defined by histologic grading of vascular thrombosis and tumor necrosis. RESULTS: After thrombogenic treatment, half of the HT-1080 xenograft-bearing animals showed only minor (=nonresponder) whereas the other half showed extensive tumor thrombosis (=responders). For responders, a significant decrease of DeltaR2* and VVF was observed compared with the control group (DeltaR2*: controls: 16 +/- 1 s-1 vs. responder: 4 +/- 2 s-1; P < 0.001) whereas DeltaR2* and VVF remained nearly unchanged for nonresponders (DeltaR2*: nonresponder 14 +/- 2 s-1). VVF and DeltaR2* values correlated inversely with the histologic grading of vascular thrombosis and tumor necrosis (VVF: r = -0.8; DeltaR2*: r = -0.71; P < 0.01). CONCLUSION: USPIO-enhanced MRI allows a noninvasive, early assessment of treatment efficacy of thrombogenic vascular-targeting agents.

R2 and R2* mapping for sensing cell-bound superparamagnetic nanoparticles: in vitro and murine in vivo testing.

PURPOSE: To prospectively determine the cellular iron uptake by using R2 and R2* mapping with multiecho readout gradient-echo and spin-echo sequences. MATERIALS AND METHODS: All experiments were approved by the institutional animal care committee. Lung carcinoma cells were lipofected with superparamagnetic iron oxides (SPIOs). Agarose gel phantoms containing (a) 1 x 10(5) CCL-185 cells per milliliter of agarose gel with increasing SPIO load (0.01-5.00 mg of iron per milliliter in the medium), (b) different amounts (5.0 x 10(3) to 2.5 x 10(5) cells per milliliter of agarose gel) of identically loaded cells, and (c) free (non-cell-bound) SPIOs at the iron concentrations described for (b) were analyzed with 3.0-T R2 and R2* relaxometry. Iron uptake was analyzed with light microscopy, quantified with atomic emission spectroscopy (AES), and compared with MR data. For in vivo relaxometry, agarose gel pellets containing SPIO-labeled cells, free SPIO, unlabeled control cells, and pure agarose gel were injected into three nude mice each. Linear and nonlinear regression analyses were performed. RESULTS: Light microscopy and AES revealed efficient SPIO particle uptake (mean uptake: 0.22 pg of iron per cell +/- 0.1 [standard deviation] for unlabeled cells, 31.17 pg of iron per cell +/- 4.63 for cells incubated with 0.5 mg/mL iron). R2 and R2* values were linearly correlated with cellular iron load, number of iron-loaded cells, and content of freely dissolved iron (r(2) range, 0.92-0.99; P < .001). For cell-bound SPIO, R2* effects were significantly greater than R2 effects (P < .01); for free SPIO, R2 and R2* effects were similar. In vivo relaxometry enabled accurate prediction of the number of labeled cells. R2' (R2* - R2) mapping enabled differentiation between cell-bound and free iron in vitro and in vivo. CONCLUSION: Quantitative R2 and R2* mapping enables noninvasive estimations of cellular iron load and number of iron-labeled cells. Cell-bound SPIOs can be differentiated from free SPIOs with R2' imaging.

Antiangiogenic tumor treatment: early noninvasive monitoring with USPIO-enhanced MR imaging in mice.

PURPOSE: To prospectively investigate steady-state blood volume measurements for early quantitative monitoring of antiangiogenic treatment with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: The institutional animal care committee approved all experiments. HT-1080 fibrosarcoma-bearing nude mice were injected with a thrombogenic vascular targeting agent (VTA) (11 nude mice, 20 tumors) or saline (12 nude mice, 20 tumors). USPIO-enhanced (SH U 555C) MR imaging was performed after the VTA was administered. USPIO-induced changes in tissue R2* (DeltaR2*) were measured with a T2-weighted dual-echo echo-planar imaging sequence, and the vascular volume fraction (VVF) was calculated. Parametric DeltaR2* maps were analyzed with respect to tumor perfusion patterns. Correlative histologic analysis was performed for grading of tissue thrombosis, and tissue perfusion was quantified with fluorescent microbeads. Unpaired Student t test and Spearman nonparametric correlation coefficient were used for statistical analysis. RESULTS: The DeltaR2* values were significantly (P < .001) reduced shortly after treatment initiation (mean DeltaR2*, 0.017 msec(-1) +/- 0.0014 [standard error] in control animals vs 0.005 msec(-1) +/- 0.0007 in animals that received VTA), which was also reflected by a decrease in the VVF (2.47% +/- 0.18 vs 0.41% +/- 0.48, P < .001). Histologic analysis revealed various degrees of tumor thrombosis after VTA treatment that correlated inversely with the DeltaR2* values (r = -0.83). Moreover, tumor perfusion measurements corroborated the MR results, indicating a significant reduction in tissue perfusion after VTA treatment (mean tissue fluorescence, 570.4 arbitrary units [au] per gram +/- 27 vs 161.7 au/g +/- 17; P < .05). CONCLUSION: USPIO-enhanced MR imaging enables early monitoring of antiangiogenic treatment of tumors.

Assessment of bone marrow angiogenesis in patients with acute myeloid leukemia by using contrast-enhanced MR imaging with clinically approved iron oxides: initial experience.

PURPOSE: To prospectively assess bone marrow (BM) angiogenesis in patients with acute myeloid leukemia (AML) by using iron oxide-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was institutional ethics committee approved. Informed signed consent was obtained from each study participant. The requirement for informed consent for use of data from a reference database was waived. Eleven patients (seven women, four men; mean age, 53 years+/-4.40 [standard deviation]) with an initial diagnosis of AML were enrolled in the study and underwent T2*-weighted two-echo echo-planar MR imaging of the pelvis before and after intravenous injection of a clinically approved iron oxide blood-pool contrast agent. Six healthy control subjects (one woman, five men; mean age, 35 years+/-2.31) were examined with the same MR protocol. The iron oxide-induced change in R2* relaxation rate (DeltaR2*) was calculated, and the vascular volume fraction (VVF) of the BM was derived by dividing the DeltaR2* of the BM by the DeltaR2* of the muscle. Parametric DeltaR2* maps were calculated to visualize vessel distribution. Patients underwent BM biopsy for correlative determination of microvessel density (MVD) and vascular endothelial growth factor (VEGF). Differences in DeltaR2*, VVF, VEGF, and MVD were compared by using the Wilcoxon rank sum test. RESULTS: DeltaR2* maps showed prominent areas of highly vascularized BM in the patients with AML, whereas the control subjects had moderately vascularized BM with homogeneous vessel distribution. Quantitative analysis revealed VVF values to be significantly higher in patients with AML than in control subjects: The mean VVF in the pelvis was 9.18%+/-1.54 for patients versus 3.91%+/-0.61 for control subjects (P=.010). In accordance with MR results, MVD (P=.009) and VEGF expression (P=.017) were significantly elevated in the AML group compared with values in the control group. CONCLUSION: Iron oxide-enhanced MR imaging enables assessment of BM angiogenesis in patients with AML.

3.0 T vs. 1.5 T MR angiography: in vitro comparison of intravascular stent artifacts.

PURPOSE: To evaluate the signal characteristics of different iliac artery stents in MR angiography (MRA) at 3 T in comparison with 1.5 T. MATERIALS AND METHODS: Sixteen iliac artery stents were implanted in plastic tubes filled with a solution of Gd-DTPA and imaged at 3 T and 1.5 T using a T1-weighted 3D spoiled gradient-echo sequence. Image analysis included a subjective assessment of artifact characteristics, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) measurements in stented and unstented vessel parts, and quantitative measurements of total artifact size. RESULTS: The pattern of stent artifacts inside the stents evidently did not differ at 3 T and 1.5 T. The average total size of the artifact areas surrounding the stents was significantly larger at 3 T (P < 0.03). However, within the stented part of the vessel phantom, the signal of the lumen and its contrast to modeled surrounding tissue was significantly higher at the higher field. The mean SNR of the lumen increased from 95.5 at 1.5 T to 127.3 at 3 T, and the CNR of the vessel increased from 70.3 to 93. CONCLUSION: Assessment of the stent lumen in iliac artery stents in a phantom model is not compromised by imaging at 3 T compared to 1.5 T. The signal gain inside the stented part of the vessel lumen at higher field compensates for the higher degree of stent artifacts seen in stents made of steel or cobalt.

Cell tagging with clinically approved iron oxides: feasibility and effect of lipofection, particle size, and surface coating on labeling efficiency.

PURPOSE: To evaluate the effect of lipofection, particle size, and surface coating on labeling efficiency of mammalian cells with superparamagnetic iron oxides (SPIOs). MATERIALS AND METHODS: Institutional Review Board approval was not required. Different human cell lines (lung and breast cancer, fibrosarcoma, leukocytes) were tagged by using carboxydextran-coated SPIOs of various hydrodynamic diameters (17-65 nm) and a dextran-coated iron oxide (150 nm). Cells were incubated with increasing concentrations of iron (0.01-1.00 mg of iron [Fe] per milliliter), including or excluding a transfection medium (TM). Cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic emission spectroscopy. Cell visibility was assessed with gradient- and spin-echo magnetic resonance (MR) imaging. Effects of iron concentration in the medium and of lipofection on cellular SPIO uptake were analyzed with analysis of variance and two-tailed Student t test, respectively. RESULTS: Iron oxide uptake increased in a dose-dependent manner with higher iron concentrations in the medium. The TM significantly increased the iron load of cells (up to 2.6-fold, P < .05). For carboxydextran-coated SPIOs, larger particle size resulted in improved cellular uptake (65 nm, 4.37 microg +/- 0.08 Fe per 100 000 cells; 17 nm, 2.14 microg +/- 0.06 Fe per 100 000 cells; P < .05). Despite larger particle size, dextran-coated iron oxides did not differ from large carboxydextran-coated particles (150 nm, 3.81 microg +/- 0.46 Fe per 100 000 cells; 65 nm, 4.37 microg +/- 0.08 Fe per 100 000 cells; P > .05). As few as 10 000 cells could be detected with clinically available MR techniques by using this approach. CONCLUSION: Lipofection-based cell tagging is a simple method for efficient cell labeling with clinically approved iron oxide-based contrast agents. Large particle size and carboxydextran coating are preferable for cell tagging with endocytosis- and lipofection-based methods.

Contrast-enhanced blood-pool MR angiography with optimized iron oxides: effect of size and dose on vascular contrast enhancement in rabbits.

PURPOSE: To investigate intravascular enhancement of bolus-injectable small and ultrasmall superparamagnetic iron oxides (USPIOs) of different particle sizes and relaxivities for first-pass and blood-pool magnetic resonance (MR) angiography. MATERIALS AND METHODS: Iron oxides with different particle sizes (hydrodynamic diameters, 21, 33, 46, and 65 nm) were bolus injected intravenously at three doses (10, 20, and 40 micromol per kilogram body weight). An extracellular contrast agent (gadopentetate dimeglumine) served as a control. MR angiography was performed multiple times after intravenous injection (5-120 minutes and 24 hours later). Signal enhancement was calculated from signal intensity measurements in the abdominal aorta and renal and iliac arteries. RESULTS: Highest enhancement was seen during the first pass with all contrast agents. USPIO enhancement in the abdominal aorta increased significantly with decreasing particle size (65 nm vs 33 nm, 65 nm vs 21 nm; P <.01). CONCLUSION: The smallest iron oxide provided signal enhancement comparable with that of gadopentetate dimeglumine at 40 micromol iron per kilogram for first-pass investigations, with prolonged signal enhancement up to 25 minutes, allowing multiple measurements after injection of a single bolus.

Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment.

The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 microm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

Oligomerization of paramagnetic substrates result in signal amplification and can be used for MR imaging of molecular targets.

Magnetic resonance imaging (MRI) has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expand the capabilities MRI, we have developed a novel MR signal amplification (MRamp) strategy based on enzyme-mediated polymerization of paramagnetic substrates into oligomers of higher magnetic relaxtivity. The substrates consist of chelated gadolinium covalently bound to phenols, which then serve as electron donors during enzymatic hydrogen peroxide reduction by peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers leading to a threefold increase in atomic relaxtivity (R1/Gd). The observed relaxtivity changes are largely due to an increase in the rotational correlation time tau r of the lanthanide. Three applications of the developed system are demonstrated: (1) imaging of nanomolar amounts of an oxidoreductase (peroxidase); (2) detection of a model ligand using an enzyme-linked immunoadsorbent assay format; and (3) imaging of E-selectin on the surface of endothelial cells probed for with an anti-E-selectin-peroxidase conjugate. The development of "enzyme sensing" probes is expected to have utility for a number of applications including in vivo detection of specific molecular targets. One particular advantage of the MRamp technique is that the same paramagnetic substrate can be potentially used to identify different molecular targets by attaching enzymes to various antibodies or other target-seeking molecules.