Daniel Haddad

Adipose Mesenchymal Stromal Cell-Based Therapy for Severe Osteoarthritis of the Knee: A Phase I Dose-Escalation Trial

Osteoarthritis (OA) is the most widespread musculoskeletal disorder in adults. It leads to cartilage damage associated with subchondral bone changes and synovial inflammation, causing pain and disability. The present study aimed at evaluating the safety of a dose‐escalation protocol of intra‐articular injected adipose‐derived stromal cells (ASCs) in patients with knee OA, as well as clinical efficacy as secondary endpoint. A bicentric, uncontrolled, open phase I clinical trial was conducted in France and Germany with regulatory agency approval for ASC expansion procedure in both countries. From April 2012 to December 2013, 18 consecutive patients with symptomatic and severe knee OA were treated with a single intra‐articular injection of autologous ASCs. The study design consisted of three consecutive cohorts (six patients each) with dose escalation: low dose (2 × 106 cells), medium dose (10 × 106), and high dose (50 × 106). The primary outcome parameter was safety evaluated by recording adverse events throughout the trial, and secondary parameters were pain and function subscales of the Western Ontario and McMaster Universities Arthritis Index. After 6 months of follow‐up, the procedure was found to be safe, and no serious adverse events were reported. Four patients experienced transient knee joint pain and swelling after local injection. Interestingly, patients treated with low‐dose ASCs experienced significant improvements in pain levels and function compared with baseline. Our data suggest that the intra‐articular injection of ASCs is a safe therapeutic alternative to treat severe knee OA patients. A placebo‐controlled double‐blind phase IIb study is being initiated to assess clinical and structural efficacy.

Visualization of abscess formation in a murine thigh infection model of Staphylococcus aureus by 19F-magnetic resonance imaging (MRI).

Background During the last years, 19F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance We introduce 19F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.

Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli

Background Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.

Unwarping confocal microscopy images of bee brains by nonrigid registration to a magnetic resonance microscopy image

Confocal microscopy (CM) is a powerful image acquisition technique that is well established in many biological applications. It provides 3-D acquisition with high spatial resolution and can acquire several different channels of complementary image information. Due to the specimen extraction and preparation process, however, the shapes of imaged objects may differ considerably from their in vivo appearance. Magnetic resonance microscopy (MRM) is an evolving variant of magnetic resonance imaging, which achieves microscopic resolutions using a high magnetic field and strong magnetic gradients. Compared to CM imaging, MRM allows for in situ imaging and is virtually free of geometrical distortions. We propose to combine the advantages of both methods by unwarping CM images using a MRM reference image. Our method incorporates a sequence of image processing operators applied to the MRM image, followed by a two-stage intensity-based registration to compute a nonrigid coordinate transformation between the CM images and the MRM image. We present results obtained using CM images from the brains of 20 honey bees and a MRM image of an in situ bee brain.

Visualising the premature brain using 17.6 Tesla magnetic resonance imaging

The aim of this study was to evaluate the ability of a 17.6 Tesla magnetic resonance (MR) microscope to determine external and internal structures and three-dimensional (3D) volume rendering of premature bovine brain tissue. Two bovine embryos (Carnegie-stages 16 and 21) were examined. 3D magnetic resonance imaging (MRI) was performed with a high field MR-scanner at a field strength of 17.6 Tesla. Images with isotropic nominal resolutions up to 39.1 microm were acquired. The MR images corresponded very well with histological slices. 3D virtual models of the embryonic brain were easily produced in a relatively short time and the high field scanner provided highly detailed images of formalin fixed brain tissue. Manual segmentation and automatic volume rendering is a valuable tool for the fast generation of 3D brain models and, to some degree, can replace conventional techniques in comparative embryology.

High isotropic resolution magnetic resonance imaging of the mandibular canal at 1.5 T: a comparison of gradient and spin echo sequences

OBJECTIVES The precision of localizing the mandibular canal prior to surgical intervention depends on the achievable resolution, whereas identification of the nerve depends on the image contrast. In our study, we developed new protocols based on gradient and spin echo sequences. The results from both sequences were quantitatively compared for their agreement to identify the most suitable approach. METHODS By limiting the field of view to one side of the mandible, three-dimensional acquisitions with T1 weighted gradient and spin echo sequences were performed with 0.5 × 0.5 × 0.5 mm3 resolution within 6.5 min covering the mandibular canal from the mandibular to the mental foramen. Aliasing artefacts were suppressed by different techniques. A manual segmentation of the mandibular canal from seven healthy volunteers was performed on this section by three different observers. The surface distance of the segmented volumes was computed between both sequences as well as between the different observers as a measure of equality. RESULTS The quantitative comparison of the segmentation resulted in an average surface distance of 0.26 ± 0.05 mm between both sequences and an interobserver difference of 0.26 ± 0.08 mm for gradient and 0.29 ± 0.07 mm for spin echo data. By repeated evaluation, a difference of 0.15 ± 0.02 mm for gradient and 0.18 ± 0.03 mm for spin echo data was observed, indicating a slightly higher variability for spin echo images. CONCLUSIONS Both sequences can be used to achieve high-resolution images with good contrast and can be used for precise localization of the mandibular canal. Despite a slightly increased difference for the spin echo data, the advantage of an easy and robust setup remains.

Optimization and comparison of two practical dual-tuned birdcage configurations for quantitative assessment of articular cartilage with sodium magnetic resonance imaging

BACKGROUND In this study, two practical dual-tuned birdcage configurations for quantitative assessment of articular cartilage with sodium magnetic resonance imaging (MRI) were designed and compared. METHODS Two 1.5 T dual-tuned birdcages, a four-ring birdcage (FRB) and an alternating rungs birdcage (ARB), were built and then characterized by bench and MRI measurements. The relative uniformity (RU) and the efficiency of the coils were compared using (23)Na and (1)H B1 maps. In vivo images of a volunteer were acquired. RESULTS Bench measurements showed matching and decoupling coefficients of the quadrature channels lower than -20 dB. The RUs and 180° pulse amplitudes of the FRB/ARB were determined as: (1)H RU =94.4/74.4%, (23)Na RU =95.2/93.6%, (1)H 180° pulse amplitude =69.2/75.4 V and (23)Na 180° pulse amplitude =45.1/45.9 V. The in vivo (23)Na images acquired with the FRB show a signal-to-noise ratio (SNR) of 6 to 14 in the cartilage. CONCLUSIONS Due to its superior (1)H homogeneity and efficiency and its slightly better (23)Na homogeneity, the FRB is the overall preferred coil for the given requirements of this study. The achieved in vivo SNR is adequate for quantitative (23)Na and high resolution (1)H imaging.

Specific identification of iron oxide-labeled stem cells using magnetic field hyperthermia and MR thermometry.

Cell‐based therapies represent important novel strategies for the improved treatment of various diseases. To monitor the progress of therapy and cell migration, noninvasive imaging methods are needed. MRI represents such a modality, allowing, for example, for the tracking of cells labeled with superparamagnetic iron oxide nanoparticles. Unfortunately, the labeled cells cannot always be identified nonambiguously in the MR images. In this article, we present the combination of two different types of MR experiment to identify iron oxide‐labeled cells nonambiguously. The labeled cells appear as hypointense spots on standard T2*‐weighted MR images. Furthermore, they can be heated magnetically and subsequently identified by MR thermometry as a result of their heat dissipation. Other hypointense regions in the MR images are not heated and do not show heat dissipation. A proof‐of‐principle study was successfully performed in vitro and in vivo. The positive identification of the iron oxide‐labeled cells was demonstrated in collagen type I hydrogel phantoms and in living mice with high spatial and temporal accuracy. The motion of the in vitro samples was corrected in order to improve the specificity of the identification of labeled cells. Therefore, this method possesses the potential for cell tracking without prior knowledge about the cells, and thus allows the noninvasive monitoring of cell‐based therapies, as long as the cells contain a sufficient amount of iron oxide for detection in MR thermometry and imaging. Copyright

Dynamic MRI Assessment of Normal Knee Kinematics

Background: Knee motion is complex and has been studied intensively. The concept of an instant center of rotation postulates that the axis around which the tibia flexes and extends moves with knee flexion and extension. This concept has been contradicted by the concept of fixed axes around which the tibia and patella flex and extend during knee motion. Advanced imaging technologies help to further characterize knee motion and facilitate localization of the axis around which the tibia flexes and extends. Using dynamic magnetic resonance imaging (MRI), the purpose of this study was 1) to establish a setup for dynamic MRI knee measurements, 2) to construct a cadaver knee model and 3) to generate a mathematical algorithm to facilitate a 3-dimensional characterization of knee kinematics and calculate a defined and fixed axis around which the tibia flexes and extends. Methods: MR images were obtained using a 1.5T Magnetom Avanto MRI Scanner (Siemens Healthcare GmbH, Erlangen). A u-shaped 16 channel RF (radiofrequency) coil array with 160 mm inner diameter and a length of 180 mm was used and covered the cadaver knee on three sides. The fourth side of the knee is open to allow knee motion. A pneumatic movement device was specifically designed to generate reproducible and repetitive knee flexion and extension. The MRI sequence was synchronized with the frequency of the motion cycle. Ten cadaver knees were thawed, dissected and prepped with contrast media filled spheres. They were then fixed in the movement device and were scanned with dynamic MRI. The center of rotation was calculated using circular interpolation and the error (F) was calculated comparing the measured value at any time point with an ideal position based on the calculated flexion-extension axis (=nonlinear curve optimization). Results: Knee movement was analyzed within a motion arch of 90°. All knee specimens had intact ligamentous structures, no meniscal pathology, and no osteoarthritic changes of the cartilage surface. A fixed axis around which the tibia flexes and extends was localized in all ten specimens and the accuracy of the axis was calculated. Conclusions: This study presents a novel technique of using dynamic MRI to visualize knee kinematics ex vivo and confirms the presence of a fixed axis around the tibia flexes and extends. The passive movement device and the mathematical algorithm generates an accurate system to evaluate knee motion that will be of further assistance in characterization of physiologic biomechanics and in detection of pathological kinematics of the knee joint in vivo.

Calcium fluoride based multifunctional nanoparticles for multimodal imaging

New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF2:(Tb3+,Gd3+) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5–10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd3+ ions on the surface, the NPs reduce the MR T 1 relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about r = 0.471 mL·mg−1·s−1. Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL−1 was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF2:(Tb3+,Gd3+) NPs are suitable for medical imaging.