Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade¹. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development². The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues³. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner⁴. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques⁵. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map⁶. The resulting measurements at each voxel have been shown to strongly correlate (R²>0.9914) with data collected using dynamic mechanical analysis⁷. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.     

Non-invasive histochemistry of plant materials by magnetic resonance microscopy

Summary We have combined nuclear magnetic resonance (NMR) imaging on the microscopic scale with chemical shift selection to demonstrate the application of magnetic resonance imaging (MRI) to plant histochemistry. As an example of the method we have obtained separate images of the distribution of reserve oil and anethole in dried fennel mericarps. The technique can be employed to separately image the distribution of aromatics, carbohydrates, oils, water and possibly fatty acids in suitable plant materials.Abbreviations NMR nuclear magnetic resonance - MRI magnetic resonance imaging - COSY correlation spectroscopy - TMS tetramethylsilane     

Microbial and algal alginate gelation characterized by magnetic resonance

Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca(2+) ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin-spin T(2) magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.     

A New Brassinolide-related Steroid in the Leaves of Thea sinensis

Microscopic and dynamic mechanical properties for mixed aqueous gels of agar and gelatin have been studied. The microscopic observation showed formation of micro granules in the gels under the phase-contrast visual field. The constituent was recognized as agar by metachroma tic staining using a microspectrophotometer. Dynamic moduli of the gels were measured from 0.01 to 200 Hz by phase difference and by resonance. A minimum E' value was obtained for the mixed gel at a volume fraction of agar of 0.6. E′ of all gels and E″ of mixed gels were frequency dependent above 30 Hz.     

In vivo assessment of nodularin-induced hepatotoxicity in the rat using magnetic resonance techniques (MRI, MRS and EPR oximetry)

Acute nodularin-induced hepatotoxicity was assessed in vivo, in rats using magnetic resonance (MR) techniques, including MR imaging (MRI), MR spectroscopy (MRS), and electron paramagnetic resonance (EPR) oximetry. Nodularin is a cyclic hepatotoxin isolated from the cyanobacterium Nodularia spumigena. Three hours following the intraperitoneal (i.p.) administration of nodularin (LD50), a region of 'damage', characterized by an increase in signal intensity, was observed proximal to the porta hepatis (PH) region in T2-weighted MR images of rat liver. Image analysis of these regions of apparent 'damage' indicated a statistically significant increase in signal intensity around the PH region following nodularin administration, in comparison with controls and regions peripheral to the PH region. An increase in signal intensity was also observed proximal to the PH region in water chemical shift selective images (CSSI) of nodularin-treated rat livers, indicating that the increased signal observed by MRI is an oedematous response to the toxin. Microscopic assessment (histology and electron microscopy) and serum liver enzyme function tests (aminotransferase (ALT) and aspartate ALT (AST)) confirmed the nodularin-induced tissue injury observed by MRI. In vivo and in vitro MRS was used to detect alterations in metabolites, such as lipids, Glu+Gln, and choline, during the hepatotoxic response (2-3 h post-exposure). Biochemical assessment of perchloric acid extracts of nodularin-treated rat livers were used to confirm the MRS results. In vivo EPR oximetry was used to monitor decreasing hepatic pO2 (approximately 2-fold from controls) 2-3 h following nodularin exposure. In vivo MR techniques (MRI, MRS and EPR oximetry) are able to highlight effects that may not have been evident in single end point studies, and are ideal methods to follow tissue injury progression in longitudinally, increasing the power of a study through repeated measures, and decreasing the number of animals to perform a similar study using histological or biochemical techniques.     

Design, synthesis, physical and chemical characterisation, and biological interactions of lectin-targeted latex nanoparticles bearing Gd–DTPA chelates: an exploration of magnetic resonance molecular imaging (MRMI)

The physical and chemical parameters involved in the design and synthesis of biospecifically targeted nanoparticulate contrast media for magnetic resonance molecular imaging (MRMI) were explored in this pilot investigation. Latex nanoparticles 100, 400 and 900 nm in diameter were doubly derivatised, first with tomato lectin and then with gadolinium^III-diethylenetriamine pentaacetic acid (Gd-chelates) to target them to epithelial and endothelial glycocalyceal N-glycans and to generate contrast enhancement in magnetic resonance imaging (MRI). After intravenous injection into mice, human placental cotyledons or human Vena saphena magna, contrasty images of the vascular structures were obtained in 1.5 T MRI with spatial resolution 0.1 mm in the imaging plane and 0.6 mm in the z axis, persisting >60 min and resistant to washing out by buffer rinses. Ultrastructural analysis of the nanoparticles revealed the targeting groups at the nanoparticle surfaces and the distribution of the Gd-chelates within the nanoparticles and enabled counts for use in determining relaxivity. The relaxivity values revealed were extremely high, accounting for the strong MR signals observed. Occasionally, nanoparticles larger than 100 nm were seen in close spatial association with disrupted regions of cell membrane or of collagen fibrils in the extracellular matrix. The data suggest that 100-nm nanoparticles generate adequate contrast for MRMI and cause least disruption to endothelial cell surfaces.     

Magnetic Resonance Imaging Conditional Pacemakers: Rationale, Development and Future Directions

Pacemakers and other cardiac implantable electronic devices (CIEDs) have long been considered an absolute contraindication to magnetic resonance imaging (MRI), a crucial and growing imaging modality. In the last 20 years, protocols have been developed to allow MR scanning of CIED patients with a low complication rate. However, this practice has remained limited to a relatively small number of centers, and many pacemaker patients continue to be denied access to clinically indicated imaging. The introduction of MRI conditional pacemakers has provided a widely applicable and satisfactory solution to this problem. Here, the interactions of pacemakers with the MR environment, the results of MR scanning in patients with conventional CIEDs, the development and clinical experience with MRI conditional devices, and future directions are reviewed.     

Magnetic field direction differentially impacts the growth of different cell types

Magnetic resonance imaging (MRI) machines have horizontal or upright static magnetic field (SMF) of 0.1–3 T (Tesla) at sites of patients and operators, but the biological effects of these SMFs still remain elusive. We examined 12 different cell lines, including 5 human solid tumor cell lines, 2 human leukemia cell lines and 4 human non-cancer cell lines, as well as the Chinese hamster ovary cell line. Permanent magnets were used to provide 0.2–1 T SMFs with different magnetic field directions. We found that an upward magnetic field of 0.2–1 T could effectively reduce the cell numbers of all human solid tumor cell lines we tested, but a downward magnetic field mostly had no statistically significant effect. However, the leukemia cells in suspension, which do not have shape-induced anisotropy, were inhibited by both upward and downward magnetic fields. In contrast, the cell numbers of most non-cancer cells were not affected by magnetic fields of all directions. Moreover, the upward magnetic field inhibited GIST-T1 tumor growth in nude mice by 19.3% (p < 0.05) while the downward magnetic field did not produce significant effect. In conclusion, although still lack of mechanistical insights, our results show that different magnetic field directions produce divergent effects on cancer cell numbers as well as tumor growth in mice. This not only verified the safety of SMF exposure related to current MRI machines but also revealed the possible antitumor potential of magnetic field with an upward direction.     

Seasonal agar yield and quality inGelidium canariensis (Grunow) Seoane-Camba (Gelidiales,Rhodophyta) from Gran Canaria,Spain

The seasonal effects on yield and gel properties ofGelidium canariensis agar were investigated at two intertidal populations at the northern coast of Gran Canaria. Physical and rheological properties were measured in 1.5% w/v solutions after treatment with alkali. No significant differences were found on agar characteristics between the two sites studied. The highest yields were obtained during summer with a maximum in June (27.8%) and minimum during late autumn and winter (18–18.6%). Overall quality was highest in winter (November–January), when gel strength peaked above 850 g cm^–2. The results showed an agar of industrial quality.     

Molecular aspects of magnetic resonance imaging and spectroscopy

Magnetic resonance imaging (MRI) is a well known diagnostic tool in radiology that produces unsurpassed images of the human body, in particular of soft tissue. However, the medical community is often not aware that MRI is an important yet limited segment of magnetic resonance (MR) or nuclear magnetic resonance (NMR) as this method is called in basic science. The tremendous morphological information of MR images sometimes conceal the fact that MR signals in general contain much more information, especially on processes on the molecular level. NMR is successfully used in physics, chemistry, and biology to explore and characterize chemical reactions, molecular conformations, biochemical pathways, solid state material, and many other applications that elucidate invisible characteristics of matter and tissue. In medical applications, knowledge of the molecular background of MRI and in particular MR spectroscopy (MRS) is an inevitable basis to understand molecular phenomenon leading to macroscopic effects visible in diagnostic images or spectra. This review shall provide the necessary background to comprehend molecular aspects of magnetic resonance applications in medicine. An introduction into the physical basics aims at an understanding of some of the molecular mechanisms without extended mathematical treatment. The MR typical terminology is explained such that reading of original MR publications could be facilitated for non-MR experts. Applications in MRI and MRS are intended to illustrate the consequences of molecular effects on images and spectra.     

Influence of weak and covalent bonds on formation and hydrolysis of gelatin networks

The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.     

Influence of alkali treatment on agar from Gracilaria cornea from Yucatán, México

The effect of alkali treatments on the yield, rheological and chemical properties of agar from Gracilaria cornea growing along the Yucatáncoast were studied in order to evaluate its potential for industrial use inan attractive economic standpoint. Alkali treatment was carried out with NaOH concentrations of 0.5%, 1%, 3% and 5% in a water bath at 80, 85 and 90 °C. Agar yield, gel strength, gelling and melting temperatures, sulphate, 3,6-anhydro-galactose and ash content weredetermined. The different combinations of NaOH concentration and treatment temperature strongly influenced agar characteristics. There was a variation in the agar content for all NaOH treatments and temperature combinations, ranging between 14.5% to 22.1%. Although the yields obtained for 0.5% NaOH at all temperatures and 1% NaOH at 80 and 85°C were higher than those required by the industry, the physical and chemical characteristics of the agar were similar to those obtained fornative agar from the same species. The gel strengths, sulphate content and gelation hysteresis obtained with agar from the 1% NaOH treatment at 90 °C are in the range required by the food industry. Treatments with 3% and 5% NaOH at all temperatures improved significantly the agar quality giving higher gel strengths (974–1758 g cm ^-2) than those reported for other Gracilaria species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of agar and agarose gels for studying mechanical impedance in rice roots

Agar and agarose gels were evaluated as systems to mechanically impede roots of rice (Oryza sativa L.). Two-layer gels were used so that seedlings established in a layer of weak gel (0.35% weight/volume) and then grew downwards to encounter a treatment gel of up to 5.0% (w/v). Agarose gels were stronger than agar gels of the same concentration, reaching a maximum penetrometer resistance of 1.2 MPa at a concentration of 5.0%, compared to 0.3 MPa with agar. The 5.0% agar gel stimulated elongation of the seminal axis by 40% in seedlings of variety TN1 (compared with elongation in the 0.2% gel), but decreased it by 15% in the variety Lac 23. Although increasing agarose concentration decreased seminal axis elongation in both varieties, the seminal axis did not reach the lower layer of treatment gel when the concentration of the treatment gel was greater than 2.0%. The decreased root elongation was therefore a non-mechanical inhibition. In experiments conducted using a different batch of agarose, these inhibitory effects were not seen and strong agarose gels stimulated seminal axis elongation. It was concluded that the agar and agarose gel systems studied were unsuitable for studying the effect of mechanical impedance on the elongation of rice roots and that great care should be taken in interpreting the results of experiments using gels as a growth medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantitative evaluation of NMR and MRI methods to measure sucrose concentrations in plants

Summary Developing pea (Pisum sativum L.) seeds were chosen to evaluate the performance of various nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) methods of detecting sucrose in plants. The methods included chemical shift selective imaging (CHESS), heteronuclear correlation via¹³C-¹H coupling (HMQC), and homonuclear correlation via¹H-¹H coupling (DQF). The same experiments were also performed on sucrose phantom samples to evaluate the methods in the absence of the line broadening observed in plant systems. Using the spin echo technique for multi-slice imaging, we could discern the detailed internal structure of the intact seed with a resolution of tens of microns. The proton spin-lattice relaxation time and linewidth as a function of the age of the seed were measured to optimize the efficiency of the NMR and MR experiments. The age-dependent changes in these NMR parameters are consistent with the accumulation of insoluble starch as age increases. Both the NMR and MRI results are in accord with the results of chemical analysis, which reveal that the sucrose concentration is higher in the embryo than in the seed coat, and glucose is at low concentration throughout the seed. Of the three methods for proton observation, the enhanced version of the CHESS approach (CD-CHESS) provides the best combination of sucrose detection and water suppression. Direct observation of¹³C is preferable to indirect detection using HMQC because of water signal bleed-through in samples with large (>200 Hz) linewidths.Abbreviations CD-CHESS continuous wave decoupling chemical shift selective imaging - CHESS chemical shift selective imaging - CSI chemical shift imaging - CW continuous wave - DQF homonuclear double quantum filtering - FOV field of view - FW fresh weight - GHMQC gradient version of the heteronuclear multiple quantum coherence     

Review of strategies for MRI based reconstruction of endocavitary and interstitial applicators in brachytherapy of cervical cancer

Brachytherapy plays an essential role in the curative intent management of locally advanced cervical cancer. The introduction of the magnetic resonance (MR) as a preferred image modality and the development of new type of applicators with interstitial components have further improved its benefits.The aim of this work is to review the current status of one important aspect in the cervix cancer brachytherapy procedure, namely catheter reconstruction.MR compatible intracavitary and interstitial applicators are described. Considerations about the use of MR imaging (MRI) regarding appropriate strategies for applicator reconstruction, technical requirements, MR sequences, patient preparation and applicator commissioning are included.It is recommendable to perform the reconstruction process in the same image study employed by the physician for contouring, that is, T2 weighted (T2W) sequences. Nevertheless, a clear identification of the source path inside the catheters and the applicators is a challenge when using exclusively T2W sequences. For the intracavitary component of the implant, sometimes the catheters may be filled with some substance that produces a high intensity signal on MRI. However, this strategy is not feasible for plastic tubes or titanium needles, which, moreover, induce magnetic susceptibility artifacts. In these situations, the use of applicator libraries available in the treatment planning system (TPS) is useful, since they not only include accurate geometrical models of the intracavitary applicators, but also recent developments have made possible the implementation of the interstitial component. Another strategy to improve the reconstruction process is based on the incorporation of MR markers, such as small pellets, to be used as anchor points.Many institutions employ computed tomography (CT) as a supporting image modality. The registration of CT and MR image sets should be carefully performed, and its uncertainty previously assessed. Besides, an important research work is being carried out regarding the use of ultrasound and electromagnetic tracking technologies.     

Stability of agar in the seaweed Gracilaria eucheumatoides (Gracilariales, Rhodophyta) during postharvest storage

The status of the cell-wall polysaccharide of the red seaweed, Gracilaria eucheumatoides upon postharvest storage was assessed in this study. The yield, chemical composition, physical and textural properties of alkali-treated agar extract was determined at different time intervals within 31 months of storage at dried state after harvest. Minimal fluctuation in agar yield was observed, ranging from 22.9% to 29.0%. The gel strength of agar extracts averaged 318gcm(-2) until the third month of storage but decreased considerably thereafter. The relative viscosity and molecular weight of the extracts varied inversely with storage time. Results indicated that both physical and textural parameters of agar generally decreased with storage time, likely due to depolymerization as indicated by decrease in molecular weight. Agar extracted from seaweeds up to 3 months of storage could be considered to exhibit gel quality suitable for food applications. Prolonged storage of the seaweed harvest is not recommended.     

Quantification of red myotomal muscle volume and geometry in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis) using T1-weighted magnetic resonance imaging

T1-weighted magnetic resonance imaging (MRI) in conjunction with image and segmentation analysis (i.e., the process of digitally partitioning tissues based on specified MR image characteristics) was evaluated as a noninvasive alternative for differentiating muscle fiber types and quantifying the amounts of slow, red aerobic muscle in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis). MRI-determinations of red muscle quantity and position made for the mid-body sections of three mako sharks (73.5-110 cm fork length, FL) are in close agreement (within the 95% confidence intervals) with data obtained for the same sections by the conventional dissection method involving serial cross-sectioning and volumetric analyses, and with previously reported findings for this species. The overall distribution of salmon shark red muscle as a function of body fork length was also found to be consistent with previously acquired serial dissection data for this species; however, MR imaging revealed an anterior shift in peak red muscle cross-sectional area corresponding to an increase in body mass. Moreover, MRI facilitated visualization of the intact and anatomically correct relationship of tendon linking the red muscle and the caudal peduncle. This study thus demonstrates that MRI is effective in acquiring high-resolution three-dimensional digital data with high contrast between different fish tissue types. Relative to serial dissection, MRI allows more precise quantification of the position, volume, and other details about the types of muscle within the fish myotome, while conserving specimen structural integrity.     

Magnetic resonance imaging for the study of ovarian follicles in the mouse

Additional tools to analyze follicle development would be highly advantageous because current methods require sacrifice of animals at specific times and time-consuming sectioning of tissues for histologic analysis. Magnetic resonance imaging (MRI) may provide a less involved, faster and more cost-effective method to analyze follicles in whole ovaries. Fixed ovaries were collected at different stages of the estrus cycle and after stimulation with gonadotrophins (24 and 48 h post pregnant mares serum (PMSG), and 10 and 24 h post human chorionic gonadotrophin (hCG)) with or without administration of the contrast agent gadodiamide. The MR images were generated using a vertical-bore, 11.7 Tesla MR system. Analysis of the MR images revealed large antral follicles in fixed ovaries with the oocyte and cumulus mass identifiable within preovulatory follicles. The use of gadodiamide had no impact on the quality of MR images obtained. The fixed ovaries were paraffin embedded, sectioned, and hematoxylin stained. Follicles were counted using the MR images and the histology sections. Preovulatory follicle numbers determined using MR images were comparable to those using histology; however counts of smaller follicles were inconsistent. MRI of gonadotrophin-stimulated ovaries in situ did not reveal discernable ovarian structures. Therefore, MRI is a useful tool for studying whole fixed ovaries leaving the ovary intact for additional analyses or for selection of samples based on morphology. The MRI is also useful for identifying preovulatory follicles, although analysis of smaller follicles is not possible, and thus the potential exists for cyst analysis in mouse models of polycystic ovarian syndrome (PCOS).     

Internal structure and quality assessment of fresh truffle Tuber melanosporum by means of magnetic resonance imaging spectroscopy

Abstract

In the present article, magnetic resonance imaging spectroscopy (MRI) was used to study fresh black truffle, with the aim of elucidating the internal structure, the effects of fungal invasion, and physical changes occurring in post-harvest. MRI is a non-destructive and non-invasive analytical technique offering the almost unique opportunity of studying foodstuff while leaving it intact, without any sample preparation. The internal morphology and modifications induced by external agents (e.g. fungal invasion, diseases, dehydration) are some of the aspects addressed by means of this innovative analytical tool.     

Agar polysaccharides from Gracilaria species (Rhodophyta, Gracilariaceae)

Yield, physical and chemical properties of agar from three agarophytes species (Gracilaria gracilis, G. dura and G. bursa-pastoris) were determined. The agar yield from the three species varied significantly (P<0.01). The highest yields of agar (34.8%) and the lowest (30%) were obtained from G. bursa-pastoris and G. gracilis, respectively. Highest gel strength (630+/-15 g cm(-2)) was obtained from agar extracted from G. gracilis and lowest from G. bursa-pastoris (26+/-3.6 g cm(-2)). The values of 3,6-anhydrogalactose were similar for G. gracilis and G. dura and there were no significant differences among the species. The sulfate contents varied significantly (P<0.01) and the higher value was obtained from G. bursa-pastoris. Among the three species, G. gracilis showed superior agar quality than the other two species, hence it can be considered a good potential source for industrial use.