Measuring and Mapping Functional Properties of Extracellular Matrix (ECM)

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The extracellular matrix (ECM) has multiple functions in tissues: it provides structural support for cells, determines tissue rigidity and elasticity, acts as a reservoir of growth factors, and provides an environment for remodeling in response to developmental, physiological and pathological challenges. The multiple functions and complex composition of the ECM make it difficult to mimic its various properties. The ECM of different tissues contains similar macromolecular constituents. However, the organization of these components is significantly different depending on the architectural, mechanical and biological functions of the tissue. The ECM in various tissues exhibits tissue-specific composition. For example, in cartilage large aggrecan-hyaluronic acid complexes are enmeshed in a collagen matrix providing mechanical resistance to external load. In the brain, however, these proteoglycans do not have a strong fibrous network confining and constraining them. We are developing noninvasive in vitro methods to determine structure/function relationships of ECM components using novel MR imaging methods, which have the potential for early diagnosis of ECM disorders and diseases. We have also developed a magnetization transfer (MT) MRI method, which is capable to detect immobile protons (e.g., protons on the collagen backbone), which are not detectable by conventional proton density MRI owing to their short transverse relaxation time, T2. To visualize these invisible protons the magnetization of these molecules is transferred to the free water, which is visible by MRI. In pilot studies we compared the results obtained for the concentrations of the main cartilage constituents by our MT MRI method and high definition infrared spectroscopic (HDIR) imaging measurements made on the same samples. Our novel approach has the potential to map tissue structure and functional properties in vivo and non-invasively. The equilibrium hydration of ECM components defines its swelling and mechanical properties. To study hydration, an array of complementary techniques is required to probe a wide range of length and time scales, which are statistically representative of the heterogeneous sample. Controlled hydration or swelling using an osmotic stress technique provides a direct means of determining functional properties of the ECM. Our earlier measurements revealed the role of the collagen network in limiting the hydration of normal (healthy) cartilage and ensuring a high proteoglycan (PG) concentration in the matrix, which is essential for effective load bearing. We also demonstrated that the loss of collagen network stiffness is consistent with the degradation of cartilage observed in osteoarthritis (OA). To determine the effect of hydration on tissue properties we previously developed a tissue micro-osmometer to perform experiments in a practical and rapid manner. This instrument can measure very small changes in the amount of water absorbed by small tissue samples (< 1 microgram tissue) as a function of the equilibrium activity (vapor pressure) of the surrounding tissue water. We have developed a method for mapping the local elastic and osmotic properties of various tissue samples using the Atomic Force Microscope (AFM) together with the tissue micro-osmometer. Many of the impediments that previously hindered the use of AFM to probe soft inhomogeneous samples, particularly biological tissues, were addressed by our new approach that utilizes the precise scanning capabilities of the AFM to generate large volumes of compliance data from which the relevant elastic properties can be extracted. In conjunction with results obtained from high-resolution scattering measurements, tissue micro-osmometry, and biochemical analysis, this technique allows us to map the spatial variations in the osmotic modulus within tissue specimens. For example, knowledge of the local osmotic properties of cartilage is particularly important, given that it determines the compressive resistance of the tissue to external loads. We also made rheological measurements to determine the dynamic properties of PGs (chondroitin sulfate, hyaluronic acid, aggrecan and aggrecan-HA complex) at the macroscopic level. In the context of cartilage function in joints, the dynamic response of the constituents is particularly important because the timescale of slow joint movement is significantly different from that of rapid joint movement. For relatively slow motion of joints, the viscoelastic complex fluid nature of cartilage dominates, while in the rapid motion of joints, the elastic (gel-like) nature of cartilage becomes prevalent. Based on the years of structural studies, we developed a novel biomimetic hydrogel model of cartilage consisting of a stiff polymer matrix, made of polyvinyl alcohol (PVA) with embedded microgel particles consisting of crosslinked polyacrylic acid (PAA) microgel particles. In our model the PVA network corresponds to the collagen matrix, while the charged PAA microgel particles represent the PG assemblies. The high swelling ability of the PAA gel "prestresses" the PVA network, greatly increasing the stiffness of the composite gel. This new type of gel is conceptually different from conventional filler-reinforced networks. In the PVA/PAA system the swollen PAA particles inflate the PVA network. The swelling of the PAA is limited by the tensile stress developing in the PVA matrix, which increases as the gel absorbs more liquid. This behavior is opposite to the decrease of the elastic pressure observed in conventional gels. This year Julian Rey has made significant advances developing constitutive laws of the composite media to explain the functional/biomechanical behavior using both finite element methods (FEM) and analytical models. We developed a framework based on the Localization Model of rubber elasticity to describe the swelling of semi-flexible polymer networks and we applied this extended model to the synthetic hydrogel material used to model cartilage behavior. We studied the effects of different factors corresponding to changes occurring in age- and disease-related alterations of cartilage. We have demonstrated that the osmotic and mechanical behaviors of our biomimetic model systems reproduce the properties of healthy and osteoarthritic human cartilage remarkably well. To better understand the role of the two primary components, the network polymer, and the embedded particles, we performed molecular dynamics (MD) simulations to generate a wide range of gel-like structures from compact to open gels. We also used MD to investigate the origin of the physical and chemical interactions among ECM components. We have developed a model to describe the self-assembly of aggrecan bottlebrushes. We identified a hierarchical network structure where the flat sheet behavior that emerges at large length scales coexists with crumpling conformations at intermediate length scales, a characteristic feature found in aggrecan solutions. Recently, we have studied structure formation in hyaluronic acid (HA) solutions. HA is a major constituent of the ECM, and is particularly prominent in the brain. Our SANS measurements made on HA solutions in near physiological conditions revealed a hierarchical organization composed of large clusters of size greater than several hundred nanometers. A correlation peak was observed in the SANS spectra corresponding to a size of about 80 nm. We found that the SANS response contains an osmotic component that is in good agreement with the scattering intensity estimated from independent osmotic pressure measurements. We are continuing to apply knowledge acquired through studying cartilage ECM components to better understand structure/function relationships in brain ECM, about which much less is known, despite its critical importance as a mechanical scaffolding for cells, while serving as medium through which charge, mass, momentum, and energy are all transported. These processes underlie or mediate virtually all physiological functions.