Our goal is to understand fundamental relationships between function and structure in soft tissues, in “engineered” tissue constructs, and in tissue analogues. Specifically, we study how the microstructure, hierarchical organization, composition, and material properties of a tissue affect its normal biological function and dysfunction. In adopting a physical perspective to address our research questions, we use and/or develop quantitative methods (e.g., physical measurements and mathematical models) to interrogate biological and physical model systems. Primarily, we use water to probe both equilibrium and dynamic interactions occurring among tissue constituents. To determine the equilibrium osmo-mechanical properties of well-defined model systems, we systematically vary the systems’ water content and ionic composition. To probe tissue structure and dynamics, we employ small-angle x-ray and neutron scattering, static and dynamic light scattering, and magnetic resonance imaging (MRI). We also try to understand how changes in tissue microstructure affect essential transport processes (e.g., mass, charge, and momentum). MRI methods have provided the most direct noninvasive means to characterize these transport processes in vivo and have been successful in both characterizing normal tissue microstructure and following changes in development, degeneration, and aging. We also try to translate our quantitative experimental methodologies from bench to bedside.
Imaging water diffusion in the brain and other soft tissues
Basser, Pierpaoli, Komlosh, Nevo, Jones; in collaboration with Aldroubi, Assaf, Barnett, Cohen, Freidlin, Jarisch, Marenco, Pajevic, Sen
We are continuing to develop novel water displacement magnetic resonance methods that allow us to both probe new features of tissue microstructure and assess and diagnose neurological and developmental disorders. Diffusion Tensor MRI (DT-MRI) is the most mature such technology. With it, we measure a diffusion tensor of water, D, on a pixel-by-pixel basis within tissue, both noninvasively and in vivo. Information derived from D includes the local fiber-tract orientation, the mean-squared distance that water molecules diffuse in any given direction, the orientationally averaged mean diffusivity, and other intrinsic scalar invariant quantities that are independent of the laboratory coordinate system. The scalar parameters behave like quantitative histological “stains” that are “developed” without requiring exogenous contrast agents or dyes and are useful in characterizing tissue microstructure and its physiologic state or developmental status. The bulk or orientationally averaged diffusivity has been the most successful imaging parameter proposed to date for identifying ischemic tissue regions in the brain during acute stroke. Measures of diffusion anisotropy are useful in identifying white matter degeneration (e.g., Wallerian degeneration) associated with chronic stroke. DT-MRI also provides new information that can be used to follow early developmental changes occurring in cortical gray and white matter, changes that cannot be detected with other imaging methods.
We developed new ways to use MR water diffusion data to perform “in vivo virtual dissections” of the brain, i.e., detailed structural anatomic studies of the brain that previously could be performed only ex vivo by using laborious, invasive histological methods. One approach, pioneered by Sinisa Pajevic and Carlo Pierpaoli and independently by Derek Jones, involves MR images that encode nerve fiber orientation by color, an approach that has allowed us to identify the main association, projection, and commissural white matter pathways in the brain and even to differentiate white matter pathways with similar structure and composition but different spatial orientations. To assess anatomical connectivity between functional brain regions, “DT-MRI fiber tractography” can trace out or track nerve fiber tract trajectories by following the direction along which the apparent diffusivity is a maximum. Nonetheless, while DT-MRI tractography is highly reliable in depicting coherently organized primary white matter pathways, artifactual tracts can be generated in white matter pathways with a more complex fiber topology (e.g., that cross, merge, branch). We are thus assessing the reliability of each computed tract by using the point-wise assessment of streamline tractography attributes (PASTA) approach. Using other MR data and more detailed models of water diffusion in tissue, we are also developing approaches to overcome DT-MRI’s shortcoming. In particular, a new modeling and experimental framework that describes water diffusion in white matter enables us to distinguish between water diffusing within the intra-axonal space and the extra-axonal compartments. Developed with Yaniv Assaf, the approach improves the angular resolution in tract tracing and allows us to resolve the orientation of fibers that cross. Also with Yaniv Assaf, Michal Komlosh, and Raisa Freidlin, we applied MR sequences developed originally for applications in materials sciences to probe the correlations between displacements of individual spins within tissue. Along with our more detailed mathematical models of tissue microstructure and morphology, we are able to infer more structural information about gray matter than DT-MRI can provide. With Pabitra Sen, we are developing mathematical models of water diffusion in “packs of cylinders” that resemble bundles of fibrous soft tissues, allowing us to relate the measured MR signal to important microstructural features of tissue.
In general, clinical and biological applications of DT-MRI (and of other novel displacement MRI methods) require new mathematical, statistical, and image-sciences concepts and tools in order to analyze novel multidimensional data sets. Akram Aldroubi and Sinisa Pajevic have developed algorithms for obtaining a continuous, smooth approximation to the discrete, noisy, measured diffusion tensor field data, allowing us to reduce noise and follow fibers more reliably. We also derived a new Gaussian distribution for tensor-valued random variables to establish a criterion for designing “optimal” DT-MRI experiments and have devised nonparametric empirical methods (e.g., the Bootstrap) for determining features of the statistical distribution of experimental DT-MRI data, methods that are enabling us to apply powerful statistical hypothesis tests to a wide range of important biological and clinical questions that previously could be examined only with ad hoc methods.
Assaf Y, Freidlin RZ, Rohde GK, Basser PJ. New modeling and experimental framework to characterize hindered and restricted water diffusion in brain white matter. Magn Reson Med 2004;52:965-978.
Basser PJ, Pajevic S. A normal distribution for tensor-valued random variables: applications to diffusion tensor MRI. IEEE Trans Med Imaging 2003;22:785-794.
Jones DK, Basser PJ. “Squashing peanuts and smashing pumpkins”: how noise distorts diffusion-weighted MR data. Magn Reson Med 2004;52:979-993.
Jones DK, Travis AR, Eden G, Pierpaoli C, Basser PJ. PASTA: pointwise assessment of streamline tractography attributes. Magn Reson Med 2005;53:1462-1467.
Sen PN, Basser PJ. Modeling diffusion in white matter in the brain: a composite porous medium. Magn Reson Imaging 2005;23:215-220.
MRI study of normal brain development
Pierpaoli, Basser, Chang; in collaboration with Pajevic, Rohde
Many obstacles have impeded developments in neuroimaging research into childhood brain disorders, including difficulties in obtaining data from children given their limited ability to comply with procedures; the lack of an adequate normative database with which to characterize normal brain development and against which to identify and measure aberrant brain development; the lack of adequate image analysis tools for characterizing developmental brain changes; and the lack of adequate means for disseminating and sharing data analytic tools. NICHD, NIMH, NINDS, and NIDA are therefore jointly sponsoring a multicenter consortium to study brain development in normal, healthy children and adolescents, enrolling approximately 500 children at several clinical centers around the United States. The children range from infancy to young adulthood and will be seen at different times over a six-year period. MRIs of the brain are being performed and imaging findings will be related to the results of standardized neuropsychological tests. In addition to facilitating the study of normal human brain development and providing representative and reliable normal control data for studies of children with brain disorders and diseases, the study aids in developing novel pediatric neuroimaging tools and methodologies. More information about the project can be found at the “MRI Study of Normal Brain Development” Web site at http://www.brain-child.org/.
As a data processing center for the multicenter consortium, our group processes and analyzes all DT-MRI data acquired by the various clinical centers during the course of the study. Gustavo Rohde has developed methods to warp and register multidimensional diffusion-weighted image data sets obtained from children, both to correct for distortion attributable to eddy currents and motion artifacts and to permit the data to be mapped to a common template. He found that image registration itself can alter the statistical properties of image data sets and therefore must be accounted for in subsequent statistical analysis. Lin-Chin Chang has developed several algorithms that allow DT-MRI data to be imported from the various clinical centers and has checked for acquisition artifacts. Sinisa Pajevic has been exploring the properties of a new probability distribution describing the variability of diffusion tensor data, an approach that is useful in designing optimal DTI experiments and that can be used to obtain parametric statistical distributions of important tensor-derived quantities useful in pediatric imaging applications. In general, a data processing and analysis pipeline is undergoing development and implementation to support quantitative clinical DT-MRI studies.
Basser PJ. Clinical diffusion tensor MRI. In: Edelman RR, Hesselink J, Zlatkin M, Crues J, eds. Clinical Magnetic Resonance Imaging, 3rd edition. Philadelphia: Saunders-Elsevier, 2005;320-332.
Chang L-C, Jones DK, Pierpaoli C. RESTORE: robust estimation of tensors by outlier rejection. Magn Reson Med 2005;53:1088-1095.
Rohde GK, Barnett AS, Basser PJ, Marenco S, Pierpaoli C. Comprehensive approach for correction of motion and distortion in diffusion-weighted MRI. Magn Reson Med 2004;51:103-114.
Rohde GK, Barnett AS, Basser PJ, Pierpaoli C. Estimating intensity variance due to noise in registered images: applications to diffusion tensor MRI. Neuroimage 2005;26:673-684.
Biopolymer physics: water-ion-biopolymer interactions
Horkay, Basser; in collaboration with Geissler, Hecht, Tasaki
To help us understand the nature of physical/chemical interactions taking place in biomolecules and biomolecular assemblies in living systems, we have developed an experimental approach for studying the assemblies’ structure (morphology) and thermodynamic properties as a function of the length scale (i.e., spatial resolution) that is probed. The methodology combines low-resolution macroscopic osmotic swelling pressure measurements and various high-resolution scattering experiments. Macroscopic swelling pressure measurements probe the system in the coarse length-scale regime, providing information about the overall thermodynamic response. Small-angle neutron scattering (SANS), static light scattering (SLS), and dynamic light scattering (DLS) collectively provide information about the size of structural elements and the elements’ respective contribution to macroscopic osmotic properties. Combining measurements from all these techniques allows us (1) to separate the scattering intensity caused by thermodynamic concentration fluctuations from large static superstructures (e.g., aggregates) and (2) to determine the length scales of fluctuations that give rise to the macroscopic thermodynamic properties. This combined thermodynamic and structural information cannot be obtained by other techniques.
Specifically, we have applied our experimental approach to studying the interactive effects of multivalent cations, particularly calcium, on the structure and morphology of various biomolecules. Divalent cations are ubiquitous in the biological milieu, yet existing theories do not adequately explain their effect on and interactions with charged polymers or biomolecules. Moreover, it is difficult to perform experiments to study these interactions in solution; above a low concentration threshold, multivalent cations generally cause phase separation or precipitation of charged molecules. Given that macroscopic phase separation does not occur in cross-linked gels, we have cross-linked our biopolymers, extending the range of ion concentrations over which the system remains stable and can be studied. In pilot studies, we have used the new method to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and of DNA. Concentrated DNA solutions and novel DNA gels have never before been investigated with SANS in conjunction with osmotic measurements. The proposed method also provides a unique framework for analyzing the osmotic and scattering behavior of other biomolecular systems.
Geissler E, Hecht AM, Rochas C, Horkay F, Basser PJ. Light, small angle neutron and X-ray scattering from gels. Macromol Symp 2005;227:27-38.
Horkay F, Basser PJ. Osmotic observations on chemically cross-linked DNA gels in physiological salt solutions. Biomacromolecules 2004;5:232-237.
Horkay F, Basser PJ, Hecht AM, Geissler E. Structural investigations of a neutralized polyelectrolyte gel and an associating neutral hydrogel. Polymer 2005;46:4242-4247.
Morfin I, Horkay F, Basser PJ, Bley F, Hecht AM, Rochas P, Geissler E. Adsorption of divalent cations on DNA. Biophys J 2004;87:2897-2904.
Functional properties of extracellular matrix
Horkay, Basser; in collaboration with Washburn
The swelling behavior of cartilage is exquisitely sensitive to the biochemical and microstructural changes occurring in development, disease, degeneration, and aging. Knowledge of the mechanisms affecting cartilage hydration (water uptake) is also essential to understanding and predicting cartilage’s load-bearing and lubricating ability, given that the osmotic properties of cartilage’s constituents are what primarily allow cartilage to resist applied loads and express or imbibe fluid.
We have previously shown that controlled hydration or swelling of cartilage can provide a means of determining the functional properties of its extracellular matrix (ECM), thereby specifically permitting measurement of important physical/chemical properties of the collagen network and proteoglycans (PG) in cartilage independently within the ECM. Such measurement involves (1) modeling the ECM as a composite material consisting of two distinct phases: a collagen network and a proteoglycan solution trapped within it; (2) applying various known levels of equilibrium osmotic stress; and (3) using physical-chemical principles and additional experiments to determine a “pressure-volume” relationship for both the collagen and the PG phases independently. In pilot studies, we used this measurement approach to determine pressure-volume curves for the collagen network and the PG phases in native and trypsin-treated normal human cartilage specimen as well as in cartilage specimens from osteoarthritic (OA) joints. In both normal and trypsin-treated specimens, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlight the role of the collagen network in limiting normal cartilage hydration and in ensuring a high PG concentration in the matrix, which are both essential for effective load bearing in cartilage and lubrication but are lost in OA. The data also suggest that the loss of collagen network stiffness, and not the loss or modification of PGs, may be the incipient event leading to the subsequent disintegration of cartilage observed in OA.
One shortcoming of our measurement approach is that analysis of a single cartilage specimen requires several days, making it unsuitable for routine pathological analysis or tissue engineering applications. In addition, the approach requires a significant amount of tissue for osmotic titration experiments. Therefore, we recently developed a new tissue micro-osmometer to perform measurement experiments practically and rapidly. The instrument can measure minute amounts of water absorbed by small tissue samples (less than 1 microgram) as a function of the equilibrium activity (pressure) of the surrounding water vapor. A quartz crystal detects the water uptake of a specimen attached to its surface. The high sensitivity of the instrument’s resonance frequency to small changes in the amount of adsorbed water allows us to measure precisely the water mass uptake of the tissue specimen. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. To illustrate the applicability of the new apparatus, we recently measured the swelling pressure of a tissue-engineered cartilage specimen.
The micro-osmometer will eventually permit us to obtain simultaneously a profile of the osmotic compressibility or stiffness of several cartilage specimens as a function of depth from the articular surface to the bone interface. It will also allow us to quantify the contributions of individual components of ECM (such as aggrecan, hyaluronic acid, and collagen) to total osmotic pressure. Moreover, it should allow us to assess the osmotic compatibility and mechanical integrity of developing tissues and of tissue-engineered cartilage (or ECM) for improved integration following implantation.
Basser PJ, Horkay F. Toward a constitutive law of cartilage: a polymer physics perspective. Macromol Symp 2005;227:53-64.
Horkay F, Horkayne-Szakaly I, Basser PJ. Measurement of the osmotic properties of thin polymer films and biological tissue samples. Biomacromolecules 2005;6:988-993.
Michelman-Ribeiro A, Boukari H, Nossal R, Horkay F. Fluorescence correlation spectroscopy study of probe diffusion in poly(vinyl alcohol) solutions and gels. Macromol Symp 2005;227:221-230.
Sheng LG, Bencherif S, Antonucci J, Jones RL, Horkay F. Synthesis and characterization of poly(ethylene glycol) dimethacrylate hydrogels. Macromol Symp 2005;227:243-254.
Sheng LG, Jones RL, Washburn NR, Horkay F. Structure-property relationships of photopolymerizable poly(ethylene glycol) dimethacrylate hydrogels. Macromolecules 2005;38:2897-2902.
Collaborators
Akram Aldroubi, PhD, Vanderbilt University, Nashville, TN
Yaniv Assaf, PhD, Tel Aviv University, Tel Aviv, Israel
Alan Barnett, PhD, Clinical Brain Disorders Branch, NIMH, Bethesda, MD
Yoram Cohen, PhD, Tel Aviv University, Tel Aviv, Israel
Raisa Freidlin, MS, Center for Information Technology, NIH, Bethesda, MD
Erik Geissler, PhD, CNRS, Université Joseph Fourier de Grenoble, Grenoble, France
Mark Hallett, MD, Human Motor Control Section, MNB, NINDS, Bethesda, MD
Anne-Marie Hecht, PhD, CNRS, Université Joseph Fourier de Grenoble, Grenoble, France
Wolfram Jarisch, PhD, LifeStar LLC, Potomac, MD
Stefano Marenco, MD, Clinical Brain Disorders Branch, NIMH, Bethesda, MD
Pedro Miranda, PhD, Universidade de Lisboa, Lisbon, Portugal
Sinisa Pajevic, PhD, Center for Information Technology, NIH, Bethesda, MD
Gustavo Rohde, MS, University of Maryland, College Park, MD
Pabitra Sen, PhD, Schlumberger-Doll Research, Ridgefield, CT
Ichiji Tasaki, MD, Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD
Newell Washburn, PhD, Materials Science and Engineering Laboratory, NIST, Gaithersburg, MD
For further information, contact pjbasser@helix.nih.gov.