13th Annual Biomedical Engineering
Student Research Symposium
Friday, April 29th 9:00 am – 4:30 pm
BME Lecture Hall, MR-5 Room 1041
9:00 ~~~~~ Breakfast~~~~~
9:15: Introduction: Dr. Skalak, Elizabeth Browning
PhD Candidates
Host: Meghan Nickerson
Evaluators: Dr. Bill Walker and Dr. Tom Skalak; Colin Choi, Nikki Dinola
9:20: Karthik Ranganathen
A General Cystic Resolution Metric for Medical Ultrasound
9:52: Cassandra Morris
Magnetic Fields and the Microvasculature: An Attractive Therapeutic Option?
10:24: Lee Murfee
Proliferation and NG2 Signaling by Perivascular Cells during Microvascular Remodeling
10:56: Joshua Rychak
Engineering Efficient Adhesion of Targeted Ultrasound Contrast Agents
11:38: Bin Guo
New Aspects of Actin-Myosin Interactions
12:04-1:00 ~~~~~Lunch~~~~~
Masters Candidates and Third Year Cell and Tissue Engineering Students
Host: Rosie Mott
Evaluators: Dr. Rich Price and Dr. Brian Helmke; Drake Guenther, Liz Logsdon
1:00: Shiwei Zhou
Pre-Compensated Excitation Waveforms to Suppress Transmitted
Harmonic Generation in Capacitive Micro-Machined Ultrasonic Transducers
1:32: Tony Wirtel
Proliferation of Adipose-Derived Stem Cells on Bone Tissue Engineering Scaffolds
1:52: Elisa Ferrante
Fluid Flow and Heat Transfer Design Considerations for the Development of a Magnetically Guided Microcatheter
2:12: Elizabeth Browning
Endothelial Intercellular Junction Adaptive Signaling in Response to Hemodynamic Shear Stress
2:32: Megan Gettel
Local Delivery of Vascular Endothelial Growth Factor Enhances Hematopoietic Progenitor Cell Involvement in Microvascular Remodeling
2:52-3:05 ~~~~~Break~~~~~
Third Year Biomechanics and Imaging Students
Host: Elizabeth Browning
Evaluators: Dr. John Hossack, Dr. Brett Blackman; Yinbo Li, Brian Schmidt
3:05: David Mack
Non-Invasive Analysis of Physiological Signals (NAPS): A Passive Ballistocardiography, Respiration & Movement Monitor
3:25: Peng Hu
Fat-Suppressed CINE SSFP Coronary Angiography
3:45: Dattesh Shanbhag
Anisotropic Diffusion in Human Lungs Using 3He MR Spectroscopy
4:05: Chengbo Wang
Using Longitudinal Magnetization Decay to Measure Long-Range 3He Diffusion: Theoretical Analysis of Parameter Variations on Measurement Accuracy
4:25: ~~~~~Closing Remarks~~~~~
Otis & Mary Updike Award and First Annual Student-Elected TA Award
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session I:
PhD Candidates
A General Cystic Resolution Metric for Medical Ultrasound
Karthik Ranganathan, Biomedical Engineering
William F. Walker, Biomedical Engineering
Introduction: The evaluation of imaging performance is an essential task in the development of ultrasound systems, both to predict fundamental limits on image quality and to optimize parameters for system design. The ability to accurately predict performance enables system optimization and quantitative consideration of engineering tradeoffs early in the design process and significantly reduces the time and cost investment in system development. The most common metric used to estimate scanner performance is the beamplot, which has been adapted from RADAR and is the 1D (azimuth only) or 2D (azimuth-elevation) spatial response of a system. However, ultrasound targets are significantly different from RADAR targets and the use of beamplots to characterize ultrasound systems can sometimes be misleading. We present a metric that quantitatively characterizes the 3D performance of arbitrary, broadband systems in a way that is relevant to ultrasound imaging. We also present simulation results that illustrate the implementation and usefulness of our metric.
Methods: We characterize ultrasound systems using “cystic resolution”, which is the size of the spherical anechoic cyst/void that generates a specified contrast. In other words, when comparing two systems, the system that can generate the specified contrast with the smaller void is considered to be better. The contrast is calculated by determining the ratio of the energy of the point spread function (psf) outside the cyst to the total psf energy. The psf is the 4D response (3D space and time) of an imaging system. We are currently developing a low-cost, handheld, C-scan ultrasound system that utilizes a fully sampled 2D array interfaced to a custom integrated circuit with transmit protection, analog conditioning, and sampling and digitizing circuitry. Beamforming is implemented by complex phase rotation of I/Q data that are generated by directly sampling the received radiofrequency signal. We implemented simulations that compared our current prototype system to a conventional 1D array based system that focused using time delays. While the contrast metric can be used to compare systems, we are also using it to guide system design and parameter optimization. We investigated the impact of the signal to noise ratio, quantization in the analog to digital converter, element/channel crosstalk, f/#, and choice of apodization window on system performance.
Results: Our system exhibits poor performance when compared to the conventional system operating at the elevation focus of its 1D array. Usually, however, significant portions of tissue are imaged away from the elevation focus and, unlike conventional systems, the use of a 2D array enables us to focus dynamically in elevation. Away from the elevation focus of the conventional system, we found that our system performs comparably to the conventional system when imaging cysts of relevant sizes; an impressive result because our system reduces scanner size and cost by an order of magnitude. Our simulation results indicate that an SNR of 0 dB per channel and 10 bit precision yield adequate image quality in our system. We also determined that apodization using a Nuttall window in combination with an f/1 receive aperture deliver the best performance among the f/#s and windows that we studied.
Conclusions: We have developed a metric that can reliably estimate the 3D performance of arbitrary, broadband ultrasound systems. Application of our metric eases parameter optimization because any system can be characterized by a single scalar quantity, the cystic contrast. The goal of system design then is to simply maximize the contrast. Our simulation results demonstrate the use of the metric in characterizing, comparing, and designing ultrasound systems, and show that it enables the straightforward optimization of any parameter that affects image quality.
Magnetic Fields and the Microvasculature: An Attractive Therapeutic Option?
Cassandra E. Morris, Biomedical Engineering
Thomas C. Skalak, Biomedical Engineering
Introduction: Magnetic field therapy has recently become a widely used complimentary and/or alternative medicine for the treatment of vascular as well as musculoskeletal pathologies including soft tissue injuries. Recent studies in our laboratory have demonstrated that acute static magnetic field exposure can significantly alter microvascular tone directly, but the efficacy of acute treatment on an induced injury or the effect of chronic exposure has not been investigated. These studies were designed to investigate the influence of both acute and chronic localized static magnetic field application on the microvasculature, specifically the acute effects on edema formation and resolution in the rat hind paw in addition to the effects of chronic acute exposure on the cutaneous murine microvasculature.
Methods: Histamine (Hist) or saline (Sal) (0.1ml) was injected sub-plantar into rat hind paws and were subsequently treated with an acute SMF or sham under light isoflurane anesthesia. Paw volume was measured every 30 minutes for 3 hours and changes in paw volume were compared between SMF treated and sham treated for each treatment group. Chronic SMF exposure was facilitated by applying a small magnet or size and weight matched sham to the murine dorsal skinfold chamber for 7 days. Arteriolar and venular diameters were measured at day 0, day 4 and day 7 and changes in diameter were compared between SMF treated and sham treated tissues.
Results: SMF treatment applied immediately following histamine injection significantly decreased edema formation 40-60% at all timepoints, whereas pre-treatment or treatment at maximal swelling had no significant effect. In the murine model, SMF treated vessels showed a significantly smaller increase in venular diameter, 38% and 42% at day 4 and day 7, respectively, when compared to 60% and 90% in sham treated vessels. The enlargement of sham treated vessels could be attributed to vessel dilation, structural wall remodeling, or inflammation-induced tissue swelling and is therefore abrogated by application of the SMF.
Conclusions: These results suggest that SMF treatment can reduce acute edema formation resulting from induced inflammation as well as chronic microvascular adaptations. The clinical relevance extends to the treatment of acute soft tissue injuries such as athletic strains, as well as for chronic conditions such as arthritis or other vascular pathologies involving edema or dysregulation of microvascular structure and tone. Supported by NIH AT-00582
Proliferation and NG2 signaling by perivascular cells during MICROVASACULAR REMODELING
Walter Lee Murfee III, Biomedical Engineering
Dr. Thomas Skalak, Biomedical Engineering
Introduction: Capillary arterialization describes the process during microvascular remodeling in which capillaries acquire a smooth muscle cell (SMC) coating. The source of these new SMCs could be attributed to the differentiation of abluminally positioned perivascular cells, proliferation of upstream or downstream perivascular cells, interstitial precursor cells, and/or circulating stem cells. Thus, understanding capillary arterialization and more specifically the lineage of SMCs requires understanding the dynamics of each precursor cell type. The objective of this work is to characterize the response of perivascular cells to elevated hemodynamic stress and establish Neuron-Glia Antigen 2 (NG2) as a novel marker of activated perivascular cells along venules.
Methods: SMC contractile phenotype and proliferation were assessed along vessels in intact microvascular networks exposed to increased hemodynamic stress caused by coordinated ligation of artery/vein pairs. The spatial distribution of NG2 expression was characterized in rat mesenteric tissues, which were harvested 0, 1, 3, and 5 days post an inflammatory stimulation.
Results: Mesenteric microvessels chronically exposed to hemodynamic stress elevations out to 10 days exhibited increased coverage of Smooth Muscle (SM)-Myosin Heavy Chain (MHC) positive cells. At 2, 5, and 10 days post treatment, SM-a actin positive cell proliferation levels were not different between sham and ligated groups. In quiescent mesenteric microvascular networks, NG2 was expressed by perivascular cells along arterioles and capillaries, but not along venules greater than 20 mm. In stimulated networks, NG2 expression was observed along 30% (8/27) of network draining venules after 1 day and NG2 was expressed by 63% (33/52) of draining venules after 3 days indicating a upregulation of NG2 expression along venules. By 5 days post stimulation, the percentage of draining venules expressing NG2 returned to 25% (11/44) indicating a subsequent down regulation of the proteoglycan along larger sized venules. Preliminary results from ongoing experiments assessing the functional contribution of NG2 expression via in vivo administration of functional blocking antibody suggest that NG2 inhibition attenuates the increase in vascularized area associated with the inflammation model of microvascular remodeling.
Conclusions: In response to elevated hemodynamic stress, capillary arterialization is not attributed to proliferation of existing perivascular cells and is more likely due to differentiation of existing cells or recruitment of interstitial and/or circulating precursor cells. The novel finding that NG2 is transiently upregulated along venules during angiogenesis implicates NG2 as a marker of activated perivascular cells along venules and a potential anti-angiogenesis therapeutic target. Together these results have contributed to the basic understanding of SMC lineage and have identified a new microenvironmental signal that is important in capillary and venous remodeling during microvascular network growth.
Engineering efficient adhesion of targeted ultrasound contrast agents
Joshua Rychak, Biomedical Engineering
Klaus Ley, Biomedical Engineering
Introduction: Molecular imaging is a powerful diagnostic technique that enables visualization of pathophysiology at the molecular level. In particular, ultrasound imaging has shown promise in this field when coupled with targeted ultrasound contrast agents. These agents are typically spherical gas encapsulated particles several micrometers in diameter, and may be targeted to molecular markers of disease via targeting ligands conjugated to the outer shell of the agent. These agents have been used to detect the endothelial markers of various conditions of inflammation and tumor growth using ultrasound imaging. However, in vitro analyses have suggested that the retention of ultrasound contrast agents to the targeted endothelium is low under certain conditions, which may reduce the efficacy of targeted ultrasound imaging. The current project has investigated three mechanisms of enhancing ultrasound contrast agent adhesion to the endothelial inflammatory marker P-selectin: modification of the contrast agent structure to create micron-scale projections, modifying the kinetic properties of the targeting ligand, and using low-intensity acoustic radiation to move freely-flowing contrast agents toward the targeted surface
Methods: The ultrasound contrast agent used in these studies is composed of a gaseous core of C4F10 encapsulated by a lipid monolayer, to which targeting ligands may be attached using biotin:streptavidin chemistry. Two targeting ligands were investigated: an anti-P-selectin monoclonal antibody Rb40.34, and a P-selectin binding glycosulfopeptide. The targeting ligands on the agent surface were quantified with flow cytometry and plate spectroscopy. Contrast agent adhesion was assessed in the parallel plate flow chamber on recombinant mouse P-selectin at wall shear stresses between 0.5-3.5 dyne/cm2. A novel MATLAB-based automated tracking program was used to quantify contrast agent adhesive lifetime, flux, and velocity. Contrast agents bearing micron-scale projections were created by incubating biotinylated, spherical agents with a 10X excess of streptavidin conjugated free lipid shells, which adopted a cylindrical structure. Free shells were removed from shell-conjugated agents by repeated centrifugation. These agents were targeted to P-selectin as described above. High resolution images of the contrast agent structure were obtained using epifluorescent deconvolution microscopy. Contrast agent adhesion in the presence of acoustic radiation was examined in a P-selectin coated microcapillary flow chamber at wall shear rates between 355-1013 s-1. The cellulose microcapillary was both optically and acoustically transparent, which allowed simultaneous insonation and epifluorescent observation of the flowing contrast agents. Intravital microscopy of the mouse cremaster muscle was used to assess contrast agent adhesion to inflamed endothelium in vivo.
Results: Ultrasound contrast agents targeted with a glycosulfopeptide of rapid intrinsic on-rate exhibited significantly greater efficiency of capture to the target surface, especially under enhances wall shear stress. Similarly, agents that adhere to the target surface through targeting ligands located at the tips of micron-scale projections exhibited a greater efficiency of firm, as opposed to transient, adhesion; this is likely due to the reduction in dislodging force on the target:ligand bond complex. Low-intensity acoustic radiation force significantly increases the adhesion of ultrasound contrast agents to P-selectin, and this effect is dependent upon agent concentration, acoustic pressure, and fluid flow rate.
Conclusions: Ultrasound contrast agent adhesion efficiency may be enhanced, especially under conditions of low target density and increased wall shear stress, by use of the three techniques explored here. These strategies may increase the efficacy of targeted ultrasound imaging, and are potentially applicable to a wide variety of contrast agents and delivery vehicles.
New aspects of Actin-Myosin Interactions
Bin Guo, Biomedical Engineering
William Guilford, Ph.D, Biomedical Engineering
Introduction: We and others have used dynamic force spectroscopy to study the force-dependent kinetics of receptor-ligand bond rupture. Dynamic force spectroscopy (Evans and Richie, 1997) applies broad ranges of loads or loading rates to single intermolecular bonds while measuring the bond lifetime and resulting rupture force. We have applied this technique to actin-myosin bonds in the rigor and ADP-bound states to determine if significant changes occur in the actin binding interface upon ADP dissociation. It is thought that when myosin binds to actin, the actin-binding cleft within the myosin head closes upon binding and may close further upon ADP dissociation.
Methods: Rat skeletal muscle heavy meromyosin (HMM) was bound to a vertical surface of nitrocellulose, and touched with an actin-coated microsphere held in a laser trap to let the HMM-actin bond form. Controlled instantaneous loads (1.8-26.4 pN) or loading rates (13-1280 pN/sec) were applied to the HMM-actin bonds by stepping the trap away or withdrawing the surface from the microsphere at a constant velocity. Bond lifetimes and rupture forces were then measured.
Results: Actomyosin bond rupture force was measured over » 3 orders of magnitude range of loading rates. Interpreted within the model of Evans and Ritchie (1997) the data revealed two distinct energy barriers, “inner” and “outer”. The outer energy barrier remained unchanged between the ADP and rigor states. In contrast, the inner barrier underwent large changes both in bond separation distance and unloaded dissociation rate. In the inner barrier at high loading rate, the rupture force at ADP state was higher than at rigor state. Actomyosin bond lifetime was also measured at various instantaneous loads at both states. A surprising finding was that actomyosin was a “catch” bond, characterized by an increasing lifetime within increasing load. Under these instantaneous loads, bond lifetime was maximum at »6 pN, close to the isometric force a single myosin head exerts on the actin filament during a power stroke, suggesting that “catch” bonds are mechanokinetically tuned to their physiological applications. The maximum lifetime of myosin-ADP bonds was higher than those of rigor bonds, supporting the previous dynamic force spectroscopy data
Conclusions: We conclude that there are significant changes in the mechanical properties of the actomyosin bond between the ADP and rigor states. These changes may result from subtle conformational changes in the actin-binding cleft upon ADP dissociation. Dynamic force spectroscopy is a powerful technique for studying the actin-binding interface of myosin.
Pre-compensated Excitation Waveforms to Suppress Transmitted
Harmonic Generation in Capacitive Micro-maChined Ultrasonic Transducers
Shiwei Zhou, Biomedical Engineering
John Hossack, Biomedical Engineering
Introduction: Capacitive Micromachined Ultrasonic Transducers (cMUTs) have attracted significant interest in the medical ultrasound imaging field because of their high signal bandwidth and resultant improved image resolution. However, this type of transducer inherently produces harmonics since the electrostatic force generated in the transmit mode is approximately proportional to the square of the applied voltage signal. This drawback becomes a major obstacle in harmonic imaging applications.
Methods: Two pre-compensation methods were investigated to suppress the inherent harmonics in cMUTs using pre-distorted waveforms. The first approach relies on a measurement of the transducer’s linear transfer function – which is valid for small signal levels. Using this transfer function and a measurement of the undesired harmonic signal, a pre-distorted transmit signal was calculated to cancel the harmonic inherently generated by the transducer. The second approach involves defining a desired function (including a DC offset) and then taking the square root of this function to determine the shape of the required input function.
Results: The experiment was conducted on a single element cMUT transducer (Sensant 142-12-BS-8). Finite Element Analysis (FEA) was used to simulate the cMUT device. Using the first method, due to the lack of perfect linearity, the approach took two iteration steps to achieve a very satisfactory result. The FEA simulation yielded 18.6 dB suppression of the 2nd harmonic, while the experimental result achieved 20.7 dB harmonic reduction. Using the second approach, a 5.5 dB reduction of transmitted harmonic was obtained in both FEA simulation and experimental test.
Conclusions: The pre-compensated methods successfully reduced the transmitted harmonics in cMUTs. For pre-selected levels of DC bias and AC signal level, it achieves a reduced harmonic signal. Alternatively, for the same absolute amount of acceptable harmonic generation, it enables higher fundamental signal levels to be achieved. These methods can be employed in practice since the arbitrary function generators required for the transmitter circuit are becoming more common. Ultimately, the approaches will result in improved sensitivity when cMUTs are used for diagnostic harmonic imaging. Similar improvements will be obtained when the method is used to image contrast agents using non-linear detection methods.
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SESSION II:
Masters Candidates and Third Year Cell and Tissue Engineering Students
Proliferation of Adipose-Derived Stem
Cells on Bone Tissue Engineering Scaffolds
Anthony J. Wirtel, Biomedical Engineering
Cato T. Laurencin, M.D., Ph.D., University Professor
Lillian T. Pratt Chair of Orthopaedic Surgery
Department of Biomedical Engineering
Introduction: Approximately 800,000 bone graft procedures are conducted annually in the United States. While the gold standard of care is use of autograft bone derived from the patient, there are shortcomings including need for a second surgical procedure, limited availability and initial structural strength. Bone graft substitutes have been developed but none are simultaneously osteogenic, osteoinductive and osteoconductive. Zuk et al have demonstrated that adipose tissue is a source of multipotent stem cells inducible into lineages for osteoblasts, fibroblast and adipose cells with appropriate induction media. Our hypothesis is that adipose cells will differentiate down an osteogenic lineage on scaffolds constructed from bioerodible polymers.
Methods: Scaffolds of 85:15 PLAGA were fabricated by sintering solid microspheres. Adipose tissue was derived from human infrapatellar fat pad excised during total knee replacement surgery and was conducted in accordance with guidelines established by the University of Virginia’s Institutional Review Board. Adipose stem cells were isolated and plated at a density of 7,000/cm2 in DMEM + 10% FBS and 1% penicillin/streptomycin. Expanded adipose cells were passed and plated onto PLAGA scaffolds or TCP. For alkaline phosphatase and calcium quantification, time points of 3, 7, 14 and 21 days were selected. Controls included unseeded scaffolds or TCP in either control or induction media. All plates were fed with either control media or induction media. DNA was measured with a Pico Green assay while Alkaline phosphatase was semi-quatitatively measured through colorimetric assay (BioRad, Inc).
Results: Levels of DNA as measured with a Pico Green assay indicated that cell proliferation rates for control and induction media (and on scaffolds and TCP) were similar when compared at 3,7,14 and 21 days. Analysis for bone formation markers included calcium and alkaline phosphatase. At 14 and 21 days alkaline phosphatase production in induction wells increased compared to 3 and 7 day time points however this difference was not significant. Results for calcium levels were inconclusive, showing lower calcium for cell-seeded scaffolds in control and induction media as compared with scaffolds alone (without cells) again in control and induction media. SEM images of scaffolds showed cells attaching and spreading on scaffolds in both media types.
Conclusion: This indicates that adipose-derived stem cells will proliferate on PLAGA scaffolds. Ongoing work includes analysis of media at each time point as well as further analysis of calcium release using alizarin red.
FLUID FLOW AND HEAT TRANSFER DESIGN CONSIDERATIONS FOR THE DEVELOPMENT OF A MAGNETICALLY GUIDED MICROCATHETER
Elisa A. Ferrante, Biomedical Engineering
J. A. C. Humphrey, Biomedical Engineering
Introduction: This project investigates the characteristics of the fluid dynamics and heat transfer inside a geometrically and dynamically scaled model of a magnetically controlled microcatheter for medical applications. Experiments using a Plexiglas large-scale model of a double lumen, water-cooled microcatheter have allowed the identification of flow conditions that may lead to efficient heat transfer from the guidance coils, thus preventing them from overheating and burn out.
Methods: The flow has been analyzed with a Particle Image Velocimeter (PIV) system at three Reynolds number settings (250, 500 and 1000) and two values of spacing between the inner and outer tube ends (2 cm and 4 cm). The final data sets collected with PIV were further processed using a TSI software package consisting of Insight 4 and Tecplot. Results of average velocity and vorticity have been generated with these programs as vector and contour color plots. Computational fluid dynamics (CFD) calculations of the catheter have been generated by Dr. Luis Rosales to provide a comparison with the experimental data. CFD calculations of the heated flow have also been conducted to simulate flow behavior during the operation of the coils. An analytically-based numerical model for heat transfer calculations of the guidance system has been developed and used to provide estimates of the temperatures reached by regions of the flow within the catheter’s tip.
Results: Analysis of the results reveals that, due to the complexity of the geometry, the flow in the turning end of a concentric tube configuration with an obstruction becomes unstable at a lower value of Reynolds numbers than observed in a straight tube. At the unstable flow settings, Reynolds numbers of 500 and 1000, shearing of the flow around the corners of the ring is observed to generate small recirculating regions, or vortices, in front of and behind that structure. The presence of these vortices will impact the amount of heat transfer from the coils in an actual microcatheter. To quantify this conclusion, however, further experiments that also study the heat transfer in the system, and its effects on the catheter's flow, are required. A comparison between the CFD numerical data and the PIV experimental data shows the results are in very good agreement. Results from the analytically-based numerical model for heat transfer of the guidance system show that the current design effectively cools the guidance copper wires, providing the coolant flow rate is higher than 0.001 kg/s. However, the main concern with this design is the size of the inner tube, which at higher flow rates causes a large pressure drop across the tip of the catheter. It is recommended, therefore, that the dimensions of the inner tube, which delivers coolant to the tip of the catheter, be increased to reduce this pressure drop.
Conclusions: In conclusion, the investigation discussed here provides a detailed set of experimental data, showing and analyzing the fluid flow behavior inside the magnetically guided microcatheter. Present findings can be used to prevent the formation of poor coolant circulation regions that may adversely affect catheter performance and integrity. Another purpose of these studies was to demonstrate, by means of a numerical heat transfer model, that the current microcatheter design, upon improvement of certain parameters, can accomplish the function of delivering coolant fluid to its tip and dissipating heat generated by the magnetic guidance system during operation.
Endothelial Intercellular junction Adaptive Signaling IN response to Hemodynamic shear stress
Elizabeth A. Browning, Biomedical Engineering
Brett R. Blackman, Biomedical Engineering
Introduction: Hemodynamic shear stress patterns are spatially distinct throughout the human vasculature and it is thought that these heterogeneities in flow patterns result in regionally distinct endothelial phenotype. These localized phenotypes can contribute to the development of region-specific vascular pathological events such as atherosclerosis and tumor angiogenesis. Many proteins and protein complexes characterize vascular endothelial cells. One key complex, which is a signaling locus and is responsible for maintaining cell-cell interaction and communication is the intercellular junction (IJ), specifically interactions of VE-Cadherin (VEC) and PECAM-1 (PEC). This complex has been identified as a locus of shear stress-mediated signaling. The VEC-catenin complex remodels in response to steady shear stress and regulates vascular permeability while PEC is important in angiogenesis and leukocyte diapedesis. Each is important in vascular function and together they coordinate signaling events that may be responsible for region-specific vascular function. We focused on examining the dynamic interaction of beta catenin (bctn) and gamma catenin (gctn) with VEC and PEC and what role the interactions of these catenins play in affecting endothelial functional phenotype.
Methods: Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in M199 media (Cellgro/Media Tech) buffered with HEPES (Gibco). The media also contains 10% fetal bovine serum (Biowhitakker), 1:100 L-glutamate, Penicillin-Streptomycin (Biowhitakker), and 1:1000 Endothelial Cell Growth Supplement (ECGS) and Heparin. Cells were plated gelatin-coated plastic in wells specially designed for shearing device. Prior to shearing cells, standard growth media containing 2% Dextran was exchanged for media without Dextran. Cells were exposed to and abdominal arterial-like waveform with time-averaged shear stress of 7.5 dyn/cm2 using a cone-and-plate flow chamber with a 0.5º cone angle. Shearing was prolonged for 24, 48, or 72 hrs. Cells were lysed in either a RIPA buffer with protease inhibitors or fractionated into Triton X-100–soluble and Triton X-100-insoluble parts. Media containing 2% dextran was used during flow experiment as shearing media in order to minimize shearing velocity while retaining shear stress. It was exchanged during the course of the experiment at a rate of 4mL/min. The shearing device was enclosed within a chamber that as maintained at 5% C02–95% air in order to maintain pH.
Results: Our results illustrate a time-dependent shift in association of bctn and gctn with VEC and PEC implicating an adaptation of the signaling behavior. gtn interacts more predominantly with cytoskeleton-bound-PEC at later times whereas bctn increasingly interacts with cytoskeleton-bound-VEC. Flow shift experiments of pre-sheared cells highlight temporal adaptation of PEC tyrosine-phosphorylation which is reduced in response to flow shift after 48hrs of pre-shearing vs. after 24hrs. Ongoing work is investigating flow-mediated nuclear translocation of bctn/gctn and subsequent regulation of transcriptional activity.
Conclusions: The shear stress-mediated adaptation of IJ signaling suggests not only a key structural role for bctn and gctn but also physiological consequences of this signaling including development of vascular disease.
Local Delivery of Vascular Endothelial Growth Factor Enhances Hematopoietic Progenitor Cell Involvement in Microvascular Remodeling
Megan Gettel, Biomedical Engineering
Thomas C. Skalak, Biomedical Engineering
Introduction: Hematopoietic progenitor cells have been found not only to contribute to nonhematopoietic tissues, including muscle, liver, vasculature and skin but also to provide a supportive role in remodeling tissues. Understanding how these cells function in vivo can lead to an increased ability to manipulate their behaviors for desired therapeutic applications. A new method for the visualization of hematopoietic progenitor cells (HPCs) in vivo has been developed that offers the opportunity to study certain HPC behaviors, including homing ability and cytokine responsiveness. Chronic visualization of these cells in an intravital model can offer key insights into their behaviors on a day-to-day basis.
Methods: On Day 0, dorsal skinfold window chambers were surgically implanted on male C57/BL6 mice exposing the microcirculation of the subcutaneous tissue. On Day 1, HPCs were isolated from whole bone marrow using a negative cell selection technique where Lin+ cells in a whole bone marrow cell suspension are made denser than the desired Lin- cells, allowing their separation via buoyant density centrifugation. The cells are then labeled with the fluorescent membrane marker DiI, and injected subcutaneously surrounding the dorsal skinfold window chamber. The cells that reach the microvasculature viewing area in the chamber can then be visualized intravitally over a two week period. Vascular Endothelial Growth Factor (VEGF) beads were formed by adding 5 mg VEGF to 500 mg of 50/50 poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol), or PLGA. On Day 1, treated mice received five VEGF/PLGA beads greater than 500 mm each. The purpose of the local application of the PLGA beads was to ensure that the entire network received exposure to VEGF. Control mice received no beads. Chronic observations of the microvascular network were made over a 2 week period using both fluorescent and light microscopy.
Results and Conclusions: Mice that received local VEGF delivery via PLGA beads consistently exhibited higher numbers of hematopoietic progenitor cells associating with microvascular networks within the chamber. On Day 4, Day 7, Day 10, and Day 13 treated mice had an average of 28%, 69.6%, 264%, and 106.7% more DiI positive progenitor cells in vascularized areas of the window chamber, respectively. However, additional experimentation will be needed to confirm that this data trend is significant. It is hypothesized that VEGF might play an important role not only in the recruitment of HPCs to the dorsal skinfold window chamber, but also in maintaining the presence of these cells in areas of microvascular remodeling.
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SESSION III:
Third Year Biomechanics and Imaging Students
NON-INVASIVE ANALYSIS OF PHYSIOLOGICAL
SIGNALS (NAPS): A Passive Ballistocardiography, Respiration & MOVEMENT
MONITOR
David C. Mack, Biomedical Engineering
Robin A. Felder, Ph.D., Pathology
Thomas C. Skalak, Ph.D., Biomedical Engineering
Introduction: The ballistocardiogram (BCG) involves the recording of movements resulting from the forces generated by cardiologic contraction and relaxation. The motivation behind using BCG data stems from the ability of the BCG to further characterize myocardial function from a mechanical perspective. Additionally, ballistocardiography has additional benefits of being non-invasive so that it can be deployed to obtain measurements passively in subjects who are traditionally non-compliant [1]. A bed based cardiovascular and breathing monitor, the Non-Invasive Analysis of Physiological Signals (NAPS) system implementing ballistocardiography was designed and developed at the Medical Automation Research Center at the University of Virginia. The NAPS system relies on a highly sensitive pressure transducer connected to a compliant force-coupling pad installed on the mattress of a bed, on which the subject lies in order to acquire the data. The analog signal is filtered and amplified before being digitized by A-to-D converters. The passive nature of the NAPS system means that data can be recorded longitudinally, providing long-term data and personalized norms that can be instrumental in detecting changes in physiological parameters. The NAPS instrumentation system is also easily integrated into existing beds and chairs since it can be deployed as a simple disposable pad.
Methods: Simultaneous readings from a Limb Lead I configuration electrocardiogram (ECG), and the NAPS system, were taken from 16 subjects. Each subject participated in a series of one minute data recordings – 3 while lying on their back, 3 while lying on their side and 3 while lying on their stomach. Unlike the ECG, the NAPS system uses ballistocardiography. The corresponding mechanical result from the R-wave of the ECG is the systolic portion of the BCG. The NAPS system recorded data from two different parts of the body (upper chest at the heart level, and the abdomen just above the waist). Average heart rate for each recording was determined by calculating the average instantaneous R-R intervals for the ECG data while an automatic algorithm developed by this author was used for the NAPS system. Additionally, the subjects were asked to count their breaths during each recording. Since taken over 1 minute, the average breathing rate corresponded directly to this count. The corresponding breathing waveforms from the NAPS system were also recorded for comparison and an automatic algorithm developed by this author was used to generate the average breathing rate data.
Results: The NAPS data correlated well with both the ECG and the subject breathing counts. Average heart rate measurement comparison between the NAPS system and the ECG agreed well (R2 = 0.927, standard error of the mean (SEM) = 2.25 beats per minute, N = 140). As one might expect, when analyzed by position, data taken while the subject was lying on their back resulted in the best data with a SEM of 1.76 beats per minute. The lower accuracy of the other positions could probably be attributed to increased amplitude in the breathing component of the composite signal which could interfere with the extracted pulse waveform. Similarly, average breathing rate measurement comparison between the NAPS system and subject count yielded a R2 of 0.954 and a SEM of 0.943 breaths per minute.
Conclusions: Clearly, the NAPS system shows a strong correlation to both the ECG and the subject breathing count. The results illustrate that the NAPS system can indeed passively detect average heart and respiration rates over the course of one minute. Further refinement and improvement of the algorithms used to calculate the NAPS data could lead to even more accurate measurements.
FAT-SUPPRESSED CINE SSFP CORONARY ANGIOGRAPHY
Peng Hu, Biomedical Engineering
Craig Meyer, PhD, Biomedical Engineering
Introduction: The goal of this study was to develop a fat-suppressed cine SSFP sequence for gated, time-resolved coronary angiography. Refocused SSFP (SSFP, TrueFISP, FIESTA, Balanced FFE) sequences are widely used in cardiac imaging, because of their high SNR and excellent contrast between blood and myocardium. Cine SSFP sequences are not typically used for coronary angiography, because these sequences do not include fat suppression and the major coronary vessels are surrounded by fat. Investigators have studied gated SSFP coronary sequences that interrupt the steady-state to apply saturation pulses. The FEMR technique has been used to suppress fat in peripheral angiography. We have combined a cine SSFP sequence with FEMR to acquire 2D coronary artery movies with steady-state cardiac gating.
Methods: With steady state maintained throughout the heart cycle, the RF phase alternates between 0 and 90 degrees and the system frequency is midway between the resonance frequency of water and fat. Additionally, each phase encoding line is duplicated for the readouts after the 0 and 90 RF phases. The data acquisition was active only after the 90 RF phase, so that only fat-suppressed images were generated.
Coronary images of normal volunteers were acquired on a 1.5T Siemens Sonata scanner with a 40mT/m maximum gradient strength and 200 mT/m/s maximum slew rate. The FOV was 20 cm, the in-plane resolution was 1.56 mm, the slice thickness was 7 mm, and the flip angle was 30 degrees. The TR was 2.8 ms and 4 phase encodes were acquired per heartbeat. The resulting temporal resolution was 22.4 ms, with images reconstructed every 11.2 ms using a sliding window.
Results: We did a comparison between two images, one acquired using a standard cine SSFP sequence and one with the cine SSFP sequence combined with FEMR. The left coronary artery system is much better visualized in the fat-suppressed image. The fat-suppressed SSFP image maintains good contrast between blood and myocardium throughout the heart cycle, which helps with the visualization of smaller branches and allows a thicker slice to be used. The cine presentation allows the operator to view various coronary segments as they come into the image plane and at the cardiac phases that minimize motion blurring.
Discussion: FEMR can achieve fat suppression of approximately 85% and, at the same time, take advantage of the steady state contrast and high temporal resolution of SSFP sequence, which makes it applicable to coronary artery imaging. Although we have to discard every other readout to achieve fat suppression, we can still acquire cine coronary images during a reasonable breathhold. It is straightforward to trade off temporal resolution for higher spatial resolution.
Conclusion: Cine SSFP combined with FEMR provides SSFP contrast, good fat suppression, which makes it a promising technique for coronary artery imaging.
ANISOTROPIC DIFFUSION IN HUMAN LUNGS USING 3HE MR SPECTROSCOPY
Dattesh Shanbhag, Biomedical Engineering
Jack Knight Scott, PhD, Biomedical Engineering
Introduction: Measurement of 3He apparent diffusion coefficient (ADC) in the human lungs presents an opportunity for probing the morphology of the lung microstructure. Currently 3He diffusion weighted imaging is used to determine the ADC along a single axis by sampling the diffusion curve using only a few b-values (frequently two). As the 3He gas motion is less restricted along the axis of the airways as compared to the directions orthogonal to it, the diffusion in lungs is anisotropic [1]. However, given the time constraints for the imaging technique, an anisotropic measurement of diffusion is difficult and highly susceptible to noise due to sparse sampling of diffusion curve. In this study, we demonstrate the application of 3He magnetic resonance spectroscopy (3He-MRS) to determine global anisotropic diffusion in vivo. The method is robust because it densely samples the diffusion curve over a large range of b- values within a single, short breathold (< 7s).
Methods: All the experiments were performed on a 1.5T whole body Siemens Sonata MRI system using a helium chest coil. For each subject, 150 lung spectra were collected during a 6.40 s breathold using a non-selective 5°, 1 ms gaussian RF pulse and TE/TR = 6.2 ms/ 40.5 ms. Bipolar trapezoidal gradients were used for diffusion sensitization along the right-left (R-L), anterior-posterior (A-P) and head-foot (H-F) anatomical directions with the volunteer in feet first supine position. Forty b-values (from 54.0 s/cm2 to 0.0 s/cm2) were logarithmically sampled along each of the directions mentioned above. Ten FIDs were collected without the diffusion sensitization gradients to obtain a measure of flip angle and T1 attenuation effects. The FIDs were phase corrected and the peak areas obtained using AMARES algorithm provided in jMRUI (version 2.2, 2005). The data were corrected for flip angle dependent attenuation and T1 relaxation using the method described in [2] and fitted to a multi exponential model. The data was also fit to the geometrical model suggested in [1].
Results and Discussion: The diffusion curve in vivo does not follow a mono-exponential model. A bi-exponential model fits well to the experimental data. Models with m > 2 had very high coefficients of variation, did not improve the fit, and were consequently rejected. There is substantial flip angle and T1 related signal loss (~60%) during the entire experiment. This signal loss should be accounted for when measuring the ADC as the diffusion curve also incorporates this attenuation. This study demonstrates the anisotropic nature of diffusion in human lungs. H-F direction demonstrated the largest ADC value which suggests that this direction offers least restraint to diffusion of 3He. The A-P direction had the lowest ADC value which indicates maximum restraint to diffusion along this direction. Fitting of the data to geometrical model suggested in [1] gave values for the external lung radius in range of 280 – 330 um which is in agreement with in-vitro measurements.
Conclusion: The mono-exponential model was found to follow the diffusion curve only for b-values < 15 s/cm2. For the range of b-values (0 to 54 s/cm2) used in the experiment, the failure of mono exponential model was conspicuous. A bi-exponential model fit the experimental data well. The anisotropic diffusion in lungs was distinctly manifested. Thus 3He-MRS provides a useful probing tool of lung morphology by allowing dense sampling of diffusion curve over a large range of b-values, along three directions within a single short breathold.
References:
[1] Yablonskiy D.A., Sukstanskii A.L., Leawoods J.C., et. al, Proc Natl Acad Sci U.S.A, 99(5):3111-3116, 2002. [2] Knight-Scott J., Smith A. L. and Mugler J.P., Proc .ISMRM , pg. 1898, Sydney, April 1998.
Using Longitudinal Magnetization Decay to Measure Long-Range 3He Diffusion: Theoretical Analysis of Parameter Variations on Measurement Accuracy
Chengbo Wang, Biomedical Engineering
John Mugler III, Radiology
Introduction: Recently, a new method was introduced to measure “long-range” diffusion over a period of seconds from the decay of “tagged” longitudinal magnetization [1]. Many different parameters may influence the accuracy of such long-range diffusion measurements. The purpose of this study was to use theoretical simulations to investigate the influence of B1-inhomogeneity, signal-to-noise ratio (SNR) and the number of images on the accuracy of long-range diffusion measurements.
Methods: Our model was based on a simulated axial image of the lung. The spin density and T1 were assumed to be spatially uniform. Sinusoidal spatial modulation (tagging) of the longitudinal magnetization was simulated from the equations in reference 1. The effects of T1 decay, diffusion and the imaging RF pulses were applied to the tagged longitudinal magnetization, and Gaussian white noise was added into the resulting images. The final simulated image intensity is given by equation 1:
(Equation 1)
Different parameters were assumed to simulate. The average value, and the cosine and sine components of the image intensity were determined from the simulated series of images according to the calculation scheme described in reference 1. The fractional modulation FM at position x was determined from equation 2 [1]:
(Equation 2)
This procedure resulted in a new series of images that contained the FM values defined for each pixel. The FM values were fitted to a mono-exponential decay to determine the long-range diffusion coefficient on a pixel-by-pixel basis
To investigate the effects of these different parameters, we varied one parameter value while keeping the other parameter values constant, and then compared the simulated diffusion values with the assumed diffusion coefficient of 0.07 cm2/s. The mean and standard deviation of the simulated diffusion values were determined for each set of parameter values.
Results: Representative simulated images showing the decay of the tagged longitudinal magnetization, and the corresponding calculated diffusion coefficient map, are shown. The standard deviation of the simulated diffusion values increased with decreasing signal-to-noise ratio, and was about 10% of the mean value when the SNR of the first image in the series was 50. For an actual diffusion coefficient of 0.07 cm2/s, the simulated mean value was closest to the actual value for the maximum number of images (8). However, for substantially higher values of the actual diffusion coefficient (e.g., 0.17 cm2/s) and a given initial SNR, the mean value was more accurately predicted by using a smaller number of images (4). Also, we found that the method was very sensitive to the tagging flip angles. In particular, when the tagging flip angle was greater than 45°, the simulated diffusion values were far different than the assumed value of 0.07 cm2/s.
Conclusion: Using the longitudinal magnetization decay offers a way to measure the long-range diffusion displacement. However, the accuracy of this method is influenced greatly by several measurement parameters, particularly the tagging flip angle.
[1] Woods JC, et al. Magn Reson Med 2004;51:1002-1008.
Session I:
Host: Meghan Nickerson
Feedback Committee: Thomas Skalak, PhD
Bill Walker, PhD
Colin Choi
Nikki Dinola
Session II:
Host: Rosie Mott
Feedback Committee: Richard Price, PhD
Brian Helmke, PhD
Drake Guenther
Elizabeth Logsdon
Session III:
Host: Elizabeth Browning
Feedback Committee: John Hossack, PhD
Brett Blackman, PhD
Yinbo Li
Brian Schmidt
Organizing Committee:
Elizabeth Browning
Kristen Wieghaus
Special Thanks to:
Liesl Amos
Bob Anderson
Burt Avery
Cheryl Rembold