Jakub Wiskirchen

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability

BACKGROUND AIMS Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles.

BackgroundFor clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSCs biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.ResultsHuman MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.ConclusionsIn our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

Thrombogenicity of Various Endovascular Stent Types: An In Vitro Evaluation

PURPOSE The aim of this study was to evaluate the thrombogenicity of different peripheral stent types in a standardized in vitro model with fresh human whole blood. MATERIALS AND METHODS Different stents (N = 77; n = 7 of each of 11 types) were implanted in polyvinyl chloride tubing loops and filled with donor blood samples. After 120 minutes of blood circulation, the thrombin-antithrombin III complex (TAT) levels, beta-thromboglobulin (beta-TG) levels, and platelet counts were assessed. RESULTS After 2 hours, significant differences were seen. TAT values (+/- SD) with the investigated stents were 31 micro g/mL +/- 20 (control, no stent), 328 micro g/mL +/- 206 (Saxx stent, peripheral medium CrNi31 L), 651 micro g/mL +/- 760 (Palmaz Corinthian Stent, 316 L stainless steel, electropolished), 1,609 micro g/mL +/- 1,264 (Palmaz Corinthian Stent, 316 L stainless steel, not electropolished), 810 micro g/mL +/- 578 (Palmaz Schatz long medium stent), 569 micro g/mL +/- 347 (Smart Nitinol stent), 1,037 micro g/mL +/- 577 (Megalink peripheral stent), 543 micro g/mL +/- 487 (peripheral stent, electropolished), 1,674 micro g/mL +/- 2,057 (peripheral stent, not electropolished), 3,128 micro g/mL +/- 1,812 (SelfX Nitinol stent, polished), 5,897 micro g/mL +/- 2,380 (SelfX Nitinol stent, unpolished), and 1,458 micro g/mL +/- 887 (bridge stent). The platelet count (x1,000/ micro L +/- SD) was 218 +/- 35 (control, no stent), 188 +/- 22 (Saxx stent), 187 +/- 20 (Palmaz Corinthian stent, electropolished), 135 +/- 37 (Palmaz Corinthian stent, not electropolished), 170 +/- 24 (Palmaz Schatz stent), 180 +/- 36 (Smart Nitinol stent), 159 +/- 26 (Megalink peripheral stent), 173 +/- 17 (peripheral stent, electropolished), 133 +/- 51 (peripheral stent, not electropolished), 123 +/- 37 (SelfX Nitinol stent, polished), 52 +/- 27 (SelfX Nitinol stent, unpolished), and 130 +/- 31 (bridge stent). CONCLUSION This standardized study showed a wide range of platelet activation after stent implantation. Electropolishing clearly reduced the thrombogenicity of the stents.

PET-CT-guided interventions in the management of FDG-positive lesions in patients suffering from solid malignancies: initial experiences.

Positron emission tomography–computed tomography (PET-CT) has gained widespread acceptance as a staging investigation in the diagnostic workup of malignant tumours and may be used to visualize metabolic changes before the evolution of morphological changes. To make histology of PET findings without distinctive structural changes available for treatment decisions, we developed a protocol for multimodal image-guided interventions using an integrated PET-CT machine. We report our first experience in 12 patients admitted for staging and restaging of breast cancer, non-small cell lung cancer, cervical cancer, soft tissue sarcoma, and osteosarcoma. Patients were repositioned according to the findings in PET-CT and intervention was planned based on a subsequent single-bed PET-CT acquisition of the region concerned. The needle was introduced under CT guidance in a step-by-step technique and correct needle position in the centre of the FDG avid lesion was assured by repetition of a single-bed PET-CT acquisition before sampling. The metabolically active part of lesions was accurately targeted in all patients and representative samples were obtained in 92%. No major adverse effects occurred. We conclude that PET-CT guidance for interventions is feasible and may be promising to optimize the diagnostic yield of CT-guided interventions and to make metabolically active lesions without morphological correlate accessible to percutaneous interventions.

The effects of paclitaxel on the three phases of restenosis: smooth muscle cell proliferation, migration, and matrix formation: an in vitro study.

Purpose:We sought to evaluate the growth-modulating potential of paclitaxel on cultured human arterial smooth muscle cells depending on the administered dose Material and Methods:For all experiments human arterial smooth muscle cells (SMCs) were used. SMCs were either cultured for 5 days or for 20 days with paclitaxel (doses: 10−7 M, 10−8 M, 10−9 M). For a total period of 20 days, proliferation kinetics of the SMC were analyzed. To assess the clonogenic activity of the SMC colony-forming assays were performed. Drug- and dose-dependent cell cycle changes were analyzed by flow cytometry. The effect on cell migration was examined in a 2-chamber migration system. The effects of paclitaxel on the synthesis of tenascin were examined via immunofluorescence. Results:Depending on the dose administered, paclitaxel proved to inhibit SMC proliferation effectively when administered during the total period of 20 days. When incubated for 5 days with doses of paclitaxel ranging between 10−8 M and 10−9 M, SMCs showed clear signs of regeneration. When being incubated with 10−7 M of paclitaxel, however, SMCs reacted with a reduction in cell proliferation, a reduced clonogenic activity, and a drug-induced G2/M phase block. SMC migration was inhibited effectively as well as extracellular matrix formation. Conclusion:Paclitaxel is a potent inhibitor of SMC proliferation, SMC migration, and extracellular matrix formation in vitro, with all three phases of the restenosis process inhibited effectively.

PET/CT-guided biopsies of metabolically active bone lesions: applications and clinical impact.

PurposeIn a minority of cases a definite diagnosis and stage grouping in cancer patients is not possible based on the imaging information of PET/CT. We report our experience with percutaneous PET/CT-guided bone biopsies to histologically verify the aetiology of hypermetabolic bone lesions.MethodsWe retrospectively reviewed the data of 20 consecutive patients who underwent multimodal image-guided bone biopsies using a dedicated PET/CT system in a step-by-step technique. Technical and clinical success rates of PET/CT-guided biopsies were evaluated. Questionnaires were sent to the referring physicians to assess the impact of biopsies on patient management and to check the clinical need for PET/CT-guided biopsies.ResultsClinical indications for biopsy were to histologically verify the aetiology of metabolically active bone lesions without a morphological correlate confirming the suspicion of metastases in 15 patients, to determine the origin of suspected metastases in 3 patients and to evaluate the appropriateness of targeted therapy options in 2 patients. Biopsies were technically successful in all patients. In 19 of 20 patients a definite histological diagnosis was possible. No complications or adverse effects occurred. The result of PET/CT-guided bone biopsies determined a change of the planned treatment in overall 56% of patients, with intramodality changes, e.g. chemotherapy with palliative instead of curative intent, and intermodality changes, e.g. systemic therapy instead of surgery, in 22 and 50%, respectively.ConclusionPET/CT-guided bone biopsies are a promising alternative to conventional techniques to make metabolically active bone lesions—especially without a distinctive morphological correlate—accessible for histological verification. PET/CT-guided biopsies had a major clinical impact in patients who otherwise cannot be reliably stage grouped at the time of treatment decisions.

Isotropic High-Spatial-Resolution Contrast-enhanced 3.0-T MR Angiography in Patients Suspected of Having Renal Artery Stenosis

The purpose of this study was to prospectively evaluate the diagnostic performance of contrast material-enhanced magnetic resonance (MR) angiography performed at 3 T for assessment of renal artery stenosis (RAS) by using parallel acquisition techniques with high acceleration factors and with digital subtraction angiography (DSA) as the reference standard. The study was institutional review board approved, and written informed consent was obtained from all patients. Twenty-nine patients (18 men, 11 women; mean age, 57.1 years +/- 14.3 [standard deviation]) suspected of having RAS underwent MR angiography. Images were evaluated qualitatively and quantitatively. The interobserver variability, sensitivity, specificity, and positive and negative predictive values of 3-T MR angiography, as compared with DSA (performed in 15 patients), were calculated. All examinations yielded good or excellent image quality. The sensitivity and specificity of MR angiography in grading significant (>75%) stenosis were 94% and 96%, respectively. Owing to its high sensitivity, contrast-enhanced 3-T MR angiography can be used reliably to exclude RAS and can serve as a useful screening method in the diagnostic work-up of patients with arterial hypertension.

Comparison of Gadolinium-BOPTA and Ferucarbotran-enhanced three-dimensional T1-weighted dynamic liver magnetic resonance imaging in the same patient.

Objectives:We sought to compare signal changes using Ferucarbotran and gadobenate dimeglumine (Gd-BOPTA) in dynamic 3D T1-weighted (T1w) GRE imaging of the liver. Material and Methods:Thirty patients were prospectively included in the study. All patients underwent 2 high-field magnetic resonance (MR) examinations: first with Gd-BOPTA (Gd) and then after a mean interval of 4 days with ferucarbotran (Feru). Dynamic MRI was obtained with a 3D T1w GRE sequence (TR 6.33, TE 2.31, flip angle 20°). Contrast enhanced scans were assessed before intravenous injection of the contrast agent (precontrast), and postcontrast during the arterial phase (30 seconds), portal venous phase (60 seconds), and equilibrium phase (120 seconds). The signal intensities (SIs) of liver, spleen, aorta, and portal vein were defined by region of interest measurements. Signal intensity changes (SICs) and percentage signal intensity change (PSIC) were calculated using the formulas SIC = (SI pre − SI post)/SI pre and PSIC = SIC × 100%. Results:Positive signal enhancement was observed after intravenous injection of Feru during all dynamic measurements, whereas the mean SI values were lower compared with Gd. During the portal venous phase the mean SI of Gd was up to a factor of 2.1 higher (portal vein). The widest difference of SIC was observed during the equilibrium phase for liver parenchyma (Gd, 1.03; Feru, 0.24). The dynamic signal courses were similar for liver, portal vein and aorta. Different signal courses were obtained for the spleen. Conclusions:Feru-enhanced T1w dynamic images demonstrated significant signal increases for liver, vessels, and spleen but overall lower signal intensities than Gd-BOPTA. The dynamic signal courses of ferucarbotran were similar to that of Gd-BOPTA during ll perfusion phases except in the spleen. Thus, it may be possible to detect typical enhancement pattern of focal liver lesions with Feru-enhanced dynamic T1w MRI.

Comparison of Digital Flat-Panel Detector and Conventional Angiography Machines: Evaluation of Stent Detection Rates, Visibility Scores, and Dose-Area Products

OBJECTIVE The objective of this study was to compare the performance and radiation doses of a flat-panel detector (FPD) angiography machine with an image intensifier (II) angiography machine. MATERIALS AND METHODS Images of four nitinol stents (Sinus-SuperFlex, SMART, Luminexx, and Zilver stents) in a phantom of a human pelvis were acquired on an FPD system (Axiom Artis) and an II system (Fluorospot TOP) using the following modes: spot-film, continuous fluoroscopy (4, 7.5, 15, and 30 pulses/s), and three amplification modes. Objective stent detection rates and subjective radiopacity scores (scale: 0 [not visible] to 4 [excellent visibility]) were calculated. The radiation doses evaluated by the respective machines were compared. RESULTS Over all modes and stents, the mean objective correct stent detection rates and mean subjective radiopacity scores were 89.49% and 1.81, respectively, for the Axiom Artis and 91.00% and 2.26 for the Fluorospot TOP. The stent detection rates over all modes for the SMART and Luminexx stents were better using the Axiom Artis machine (97.61% vs 93.55% and 98.28% vs 90.41%, respectively) and those for the Sinus-SuperFlex and Zilver stents were better using the Fluorospot TOP machine (90.83% vs 83.56% and 89.29% vs 80.50%). The subjective radiopacity scores of stent visibility were worse for the Axiom Artis than the Fluorospot TOP for all stents except the Luminexx stent (mean score, 2.34 vs 2.21, respectively). The objective stent detection rates and subjective radiopacity scores improved using the spot-film mode and with raising amplification, whereas increases in the fluoroscopy pulsing frequency did not improve stent detection rates or radiopacity scores for either machine. The radiation doses at continuous fluoroscopy were approximately 90% higher for the Axiom Artis than for the Fluorospot TOP (2.60 vs 1.41 μGy/m(2) at 30 pulses/s, respectively). CONCLUSION The objective correct stent detection rates were similar for both machines with differences in detection for the respective stents. The subjective radiopacity scores were almost always better for the Fluorospot TOP machine. Also, the Axiom Artis machine generated approximately 90% higher radiation doses in fluoroscopy. For both machines, using a higher fluoroscopy pulsing frequency had no positive effect on objective correct stent detection rates or subjective radiopacity scores.

Radiopacity of Current Endovascular Stents: Evaluation in a Multiple Reader Phantom Study

PURPOSE To evaluate the radiopacity of endovascular stents based on the fluoroscopy mode in a phantom of the human pelvis. MATERIALS AND METHODS The following stents were included in this study: Medtronic AVE Bridge, Medtronic AVE Bridge X, Cordis Covered Nitinol (Covent), Guidant Dynalink, Luminexx, Guidant Megalink, Memotherm Flexx, Palmaz Medium, Palmaz-Schatz Long-Medium, Palmaz Corinthian PQ394Q and PQ294Q, SelfX, SMART without markers, SMART with radiopaque markers, Easy Wallstent. To evaluate radiopacity, images of the stents placed in four different positions (lumbosacral junction left and right, iliosacral joint left and right) of a pelvic phantom were taken at the following modes: spotfilm, continuous fluoroscopy, 15 pulses per second, 7.5 pulses per second, and 3 pulses per second. Images were presented at random to four independent readers and radiopacity scores were assessed: 0 = not visible, 1 = poor visibility, 2 = average visibility, 3 = good visibility, and 4 = very good visibility. RESULTS The Covent stent had the highest overall radiopacity score (3.25), followed by the Luminexx (3.04) and the Medtronic AVE Bridge X (2.74) stents. At the spotfilm mode, the best visible stents were the Medtronic AVE Bridge X, the Covent and the Easy Wallstent stents and at the continuous fluoroscopy mode, the Covent, the Luminexx, and the Medtronic AVE Bridge X stents. Decreasing the fluoroscopy mode went hand in hand with a reduction of the radiopacity scores of all stents. At the standard fluoroscopy mode of 7.5 pulses per second, the Covent stent was seen well or very well in 96.9%, followed by the Luminexx (76.9%), and the Medtronic AVE Bridge X (41.25%) stents. CONCLUSIONS Stent radiopacity directly depends on the fluoroscopy mode; if the pulse frequency decreased, detecting the stents became more difficult. Stent mass correlates with stent radiopacity (e.g., Cordis Covered Nitinol, Bridge X). Radiopaque markers may improve stent radiopacity dramatically (e.g., Luminexx vs Memotherm Flexx).