Fundamental Studies and Applications of Tissue Sciences

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

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Abstract

Water-ion-polymer interactions govern the molecular organization of tissue components and determine the macroscopic mechanical/osmotic properties of tissues. Well-defined model systems designed to mimic the behavior of living systems, enable the study of their physicochemical basis. Knowledge of the molecular origin of tissue properties is essential for developing successful tissue engineering/regenerative medicine strategies. Biological polymers are often composed of polyelectrolytes whose characteristics are largely determined by the nature of the water hydrating the polyelectrolyte molecules, the counter-ions present in the solution, and the interaction between the components (polyelectrolytes, water molecules and counterions). It has been demonstrated that in many systems, especially in the vicinity of ion induced phase transition, the physical response is governed by universal principles. There are various physical forces and interactions implicated in these processes, including electrostatic repulsion and attraction, hydrophobic and hydrophilic interactions, hydrogen bonding and van der Waals forces. The important role that polymer physics plays in governing the behavior of biological systems has been emphasized by many researchers, as it offers a powerful framework to identify variables required to describe certain biological phenomena. Divalent cations, particularly calcium ions (Ca+2), in the biological milieu, play a key role. In general, experiments to determine the interactions between ions and biopolymers are difficult to perform, because above a relatively low ion concentration multivalent cations cause phase separation (or precipitation) of charged macromolecules. Since macroscopic phase separation does not occur in gels, we can overcome this limitation by cross-linking these polymers, i.e., extending the range of ion concentrations over which the systems remain stable. In pilot studies, we used various new non-destructive procedures to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid (PAA), and different biopolymers such as DNA and hyaluronic acid (HA) to determine the size of the structural elements that govern the osmotic concentration fluctuations. We combined SANS and SAXS to estimate the osmotic modulus of HA in the presence of both monovalent and divalent counterions. We also studied the dynamic properties (e.g., diffusion processes) of these systems by dynamic light scattering and determined the osmotic modulus from the relaxation response. We developed an experimental procedure to determine the distribution of counter-ions around charged biopolymer molecules using anomalous small-angle X-ray scattering (ASAXS) measurements. We analyzed and compared various network elasticity models that address essential physical aspects of biopolymer systems. We are particularly interested in understanding the mechanisms of interactions of biologically important divalent cations like Ca+2 with negatively charged biopolymers. Moreover, while there is some understanding of water-ion-biopolymer interactions in low and high ionic strength regimes, there is little known about these in the intermediate ionic strength milieu which characterizes the conditions in physiological salt solutions. Because most functional biopolymers are polyelectrolytes, studying this regime is critical for understanding a myriad of biological processes. For example, understanding the underlying physical and chemical mechanisms and interactions governing extracellular matrix (ECM) properties is essential to predict tissue biomechanical behavior, such as the load bearing ability and lubrication of cartilage, which are governed by osmotic and electrostatic forces that strongly depend on tissue hydration. A basic feature of charged polymers is that their properties are affected by many factors, such as the intrinsic properties of the polymer (e.g., chain rigidity), polymer conformation, molecular mass, the concentration and valence of counterions, as well as inter- and intramolecular interactions (e.g., ion condensation, hydration, hydrogen bonding). To date, no satisfactory theoretical framework embodying the relationship between molecular/supramolecular structure, macroscopic properties and biological function of either synthetic or natural polyelectrolyte molecules has been developed. Therefore, making systematic experimental studies on well-defined model systems under well-controlled conditions is critically important for better understanding the mechanism of complex biological processes. In many cases, these model systems become the basis of novel NMR and MRI phantoms we use to improve our ability to measure and map ECM properties. We also focus on ion-polymer interactions in bottlebrush polymers comprised of densely grafted chains tethered to a polymer backbone. These molecules are essential components of various living systems. Typical examples are cartilage and viscoelastic mucin layers forming protective coatings in the lungs, orifices and digestive tracks of humans and animals. Our aim is to better understand the physicochemical behavior of bottlebrush molecules. We have systematically investigated solutions of synthetic polyelectrolyte bottlebrushes through a combination of measurements of solution properties (osmometry, neutron and dynamic light scattering, and molecular dynamics simulations). These complementary experimental and computational methods probe vastly different length and timescales, allowing for a comprehensive characterization of synthetic polyelectrolyte bottlebrush molecules and the comparison of their properties with those of corresponding linear polymers. We found that bottlebrush polymers differ strongly from their linear polymer counterparts, not only in their architecture, but also in their physical properties that are primarily controlled by the length of the main and side-chains, the grafting density, and the steric repulsion of the side-chains. In collaboration with Prof. Xia (Department of Chemistry, Stanford University) we designed and synthesized novel bottlebrush polymers and determined the physicochemical properties of these model polymer solutions (neutralized polyacrylic acid bottlebrushes) having well-controlled molecular architecture. Although several approaches have been developed to synthesize bottlebrush polymers, the synthesis of high molecular mass materials with well-controlled main- and side-chain lengths and uniform grafting density is still challenging. Many previous studies focused on the synthesis of bottlebrush block copolymers in which the chemical differences between the main- and the side-chains result in self-assembly into different morphologies (e.g., cylindrical assemblies). Much less attention has been paid to making synthetic bottlebrush polymers in which the chemical composition of the backbone and the side-chains is the same. Despite the tremendous potential of bottlebrush polymers in both materials science and biology, the effects of the charged bottlebrush topology on the physicochemical behavior of their solutions has not yet been fully elucidated. For example, the effect of charges on the conformation of bottlebrush molecules is poorly understood. The effect of ions is particularly important in the biological milieu where both mono- and divalent counter-ions are present. Elucidating these basic physical properties is of fundamental importance to fully understand the basic molecular physics underlying certain biological functions. Our structural investigations of charged bottlebrush polymers have been complemented by molecular dynamics simulations performed by Dr. Alexandros Chremos in collaboration with Dr. Jack Douglas (National Institute of Standard and Technology).