Articular cartilage is a low-friction, load-bearing tissue located at joint surfaces. The extrace... more Articular cartilage is a low-friction, load-bearing tissue located at joint surfaces. The extracellular matrix (ECM) of cartilage consists of a fibrous collagen network, which is pre-stressed by the osmotic swelling pressure exerted by negatively charged proteoglycan aggregates embedded in the collagen network. The major proteoglycan is the bottlebrush shaped aggrecan, which forms complexes with linear hyaluronic acid chains. We quantify microscopic and macroscopic changes resulting from self-assembly between aggrecan and hyaluronic acid using a complementary set of physical measurements to determine structure and interactions by combining scattering techniques, including small-angle X-ray scattering, small-angle neutron scattering, and dynamic light scattering with macroscopic osmotic pressure measurements. It is demonstrated that the osmotic pressure that defines the load bearing ability of cartilage is primarily governed by the main macromolecular components (aggrecan and collagen) of the ECM. Knowledge of the interactions between the macromolecular components of cartilage ECM is essential to understand biological function and to develop successful tissue engineering strategies for cartilage repair.
The effect of temperature (T) is studied on the swelling of model poly(vinyl acetate) (PVAc) gels... more The effect of temperature (T) is studied on the swelling of model poly(vinyl acetate) (PVAc) gels in isopropyl alcohol. The theta temperature Θ of these gels, at which the second osmotic virial coefficient A 2 vanishes, is close to that of the corresponding high molecular mass polymer solution without cross-links so that solvent quality may be defined in the same way as the corresponding precursor polymer solution. We quantified the swelling and deswelling of PVAc gels relative to their size at the theta temperature, and also determined the effect of T on the shear modulus of these gels. It was found that both swelling and deswelling data could be reduced to a universal scaling equation of the same general form as derived from renormalization group (RG) theory for flexible linear polymer chains in solutions.
Programmed À1 frameshifting (À1 PRF) is an important biological process for the modification of g... more Programmed À1 frameshifting (À1 PRF) is an important biological process for the modification of gene expression enabled by the presence of RNA secondary and tertiary structures. However, the mechanism for this process is still under active study. Here, we investigate the role of mechanical force in À1 PRF by characterizing mechanically induced folding and unfolding of RNA pseudoknots using an enhanced atomic force microscopy (AFM) based singlemolecule force spectroscopy (SMFS) assay featuring $10 ms resolution. The pioneering SMFS study of RNA psuedoknots associated with À1 PRF used a custom-built optical trap. Unexpectedly, this study indicated PRF efficiency was correlated with the presence of alternative folding pathways (rather than with average unfolding force), indicating a complex role of RNA psuedoknots in À1 PRF involving folding dynamics. Here, we found a new folding intermediate in an RNA pseudoknot associated with the sugarcane yellow leaf virus (ScYLV) that was not observed in the original optical-trapping based assay. We speculate that the shorter linkers and stiffer force probe in our AFM assay (relative to an optical trap) are the primary reasons for this enhanced state resolution. Our initial measurements of contour length and folding kinetics indicate this folding intermediate is an RNA hairpin that is part of the overall pseudoknot structure. We observed this intermediate every time the pseudoknot folds, indicating that this intermediate is obligate for folding. Overall, our results indicate that the folding dynamics of RNA pseudoknots are significantly more complex than previously observed. Because of the role of the folding dynamics of RNA pseudoknots in À1 PRF, we expect these new insights into RNA pseudoknot folding dynamics will provide a deeper understanding into the mechanisms of À1 PRF.
We investigate the conformational properties of “ideal” nanogel particles having a lattice networ... more We investigate the conformational properties of “ideal” nanogel particles having a lattice network topology by molecular dynamics simulations to quantify the influence of polymer topology on the solution properties of this type of branched molecular architecture. In particular, we calculate the mass scaling of the radius of gyration (Rg), the hydrodynamic radius, as well as the intrinsic viscosity with the variation of the degree of branching, the length of the chains between the branched points, and the average mesh size within these nanogel particles under good solvent conditions. We find competing trends between the molecular characteristics, where an increase in mesh size or degree of branching results in the emergence of particle-like characteristics, while an increase in the chain length enhances linear polymer-like characteristics. This crossover between these limiting behaviors is also apparent in our calculation of the form factor, P(q), for these structures. Specifically, ...
Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a col... more Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 μm) elasticity mapping on thin (∼12 μm), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that ...
SummaryThe most abundant cartilage proteoglycan is aggrecan, a bottlebrush shaped molecule that p... more SummaryThe most abundant cartilage proteoglycan is aggrecan, a bottlebrush shaped molecule that possesses over 100 glycosaminoglycan (chondroitin sulfate and keratan sulfate) chains. The side‐chains are linear sulfated polysaccharides that are negatively charged under physiological conditions. Aggrecan interacts with hyaluronic acid (HA) to form large aggregates. Osmotic pressure measurements and rheological measurements are used to study the static and dynamic behavior of aggrecan assemblies at the macroscopic length scales. The microscopic properties of aggrecan solutions are determined by small angle neutron scattering, and static and dynamic light scattering (SLS and DLS). In dilute solutions aggrecan forms microgels with a diffuse boundary, composed of loosely connected clusters. The osmotic pressure of the aggrecan‐HA system decreases with increasing HA content. DLS yields a relaxation rate that varies as q3, arising from internal modes in the microgel. The relaxation rate in ...
A silicon oil-filled glass capillary array is proposed as an anisotropic diffusion MRI phantom. T... more A silicon oil-filled glass capillary array is proposed as an anisotropic diffusion MRI phantom. Together with a computational/theoretical pipeline these provide a gold standard for calibrating and validating high-q diffusion MRI experiments. The phantom was used to test high angular resolution diffusion imaging (HARDI) and double pulsed-field gradient (d-PFG) MRI acquisition schemes. MRI-based predictions of microcapillary diameter using both acquisition schemes were compared with results from optical microscopy. This phantom design can be used for quality control and quality assurance purposes and for testing and validating proposed microstructure imaging experiments and the processing pipelines used to analyze them.
for an Invited Paper for the MAR06 Meeting of the American Physical Society Synthetic and Biopoly... more for an Invited Paper for the MAR06 Meeting of the American Physical Society Synthetic and Biopolymer Gels-Similarities and Difference. FERENC HORKAY Ion exchange plays a central role in a variety of physiological processes, such as nerve excitation, muscle contraction and cell locomotion. Hydrogels can be used as model systems for identifying fundamental chemical and physical interactions that govern structure formation, phase transition, etc. in biopolymer systems. Polyelectrolyte gels are particularly well-suited to study ion-polymer interactions because their structure and physical-chemical properties (charge density, crosslink density, etc) can be carefully controlled. They are sensitive to different external stimuli such as temperature, ionic composition and pH. Surprisingly few investigations have been made on polyelectrolyte gels in salt solutions containing both monovalent and multivalent cations. We have developed an experimental approach that combines small angle neutron scattering and osmotic swelling pressure measurements. The osmotic pressure exerted on a macroscopic scale is a consequence of changes occurring at a molecular level. The intensity of the neutron scattering signal, which provides structural information as a function of spatial resolution, is directly related to the osmotic pressure. We have found a striking similarity in the scattering and osmotic behavior of polyacrylic acid gels and DNA gels swollen in nearly physiological salt solutions. Addition of calcium ions to both systems causes a sudden volume change. This volume transition, which occurs when the majority of the sodium counterions are replaced by calcium ions, is reversible. Such reversibility implies that the calcium ions are not strongly bound by the polyanion, but are free to move along the polymer chain, which allows these ions to form temporary bridges between negative charges on adjacent chains. Mechanical measurements reveal that the elastic modulus is practically unchanged in the calcium-containing gels, i.e., ion bridging is qualitatively different from covalent crosslinks.
Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine, 2015
We discuss the main findings of a long-term research program exploring the consequences of sodium... more We discuss the main findings of a long-term research program exploring the consequences of sodium/calcium ion exchange on the macroscopic osmotic and elastic properties, and the microscopic structure of representative synthetic polyelectrolyte (sodium polyacrylate, (polyacrylic acid)) and biopolymer gels (DNA). A common feature of these gels is that above a threshold calcium ion concentration, they exhibit a reversible volume phase transition. At the macroscopic level, the concentration dependence of the osmotic pressure shows that calcium ions influence primarily the third-order interaction term in the Flory-Huggins model of polymer solutions. Mechanical tests reveal that the elastic modulus is practically unaffected by the presence of calcium ions, indicating that ion bridging does not create permanent cross-links. At the microscopic level, small-angle neutron scattering shows that polyacrylic acid and DNA gels exhibit qualitatively similar structural features in spite of importan...
Despite the effort of developing various nanodelivery systems, most of them suffer from undesired... more Despite the effort of developing various nanodelivery systems, most of them suffer from undesired high uptakes by the reticuloendothelial system (RES) such as liver and spleen. Herein we develop an endogenous phosphatase triggered co-assembly strategy to form tumor-specific indocyanine green (ICG)-doped nanofibers (5) for cancer theranostics. Based on coordinated intermolecular interactions, 5 significantly altered near-infrared (NIR) absorbance of ICG, which improves the critical photoacoustic and photothermal properties. The phosphatase instructed co-assembly process, as well as its theranostic capability, were successfully conducted at different levels ranging from in vitro, living cell, tissue mimic, to in vivo. Specifically, the tumor uptake of ICG was markedly increased to 15.05 +/- 3.78 %ID/g, which was 25-fold higher than that of free ICG (0.59 +/- 0.24 %ID/g) at 4 h after intravenous injection. The resulting ultra-high T/N ratios (> 15) clearly differentiated tumors from...
The pathophysiology of osteoarthritis involves cellular and biochemical processes linked to mecha... more The pathophysiology of osteoarthritis involves cellular and biochemical processes linked to mechanical stress. A better understanding of the mechanism of these processes and how they cause changes in the composition, macro- and micro-structure, and mechanical properties of cartilage is necessary for developing effective preventative and treatment strategies. In this study, elastic and osmotic swelling properties of tissue-engineered cartilage were explored
ABSTRACTIt is biologically and clinically important to understand and explain the functional prop... more ABSTRACTIt is biologically and clinically important to understand and explain the functional properties of cartilage, such as its load bearing and lubricating ability, in terms of the structure, organization, components and their interactions. Our approach tries to explain functional material properties of these tissues as arising from polymeric interactions between and among the different molecular constituents within the tissues at different hierarchical lengthscales. We treat the tissue effectively as a complex molecular composite containing highly charged polysaccharide microgels trapped within a fine collagen meshwork. We have been developing a multi-scale experimental and theoretical framework to explain key material properties of cartilage by studying those of its constituents and the interactions among them at a variety of length and time scales. We use this approach to address important biological questions. One novel application we highlight here is the use of non-invasive...
is a highly charged bottlebrush shape biological polymer found in the extracellular matrix of tis... more is a highly charged bottlebrush shape biological polymer found in the extracellular matrix of tissues. It consists of a protein backbone (∼ =400nm long), to which about 100 linear chains of negatively-charged glucosaminoglycans are attached approximately 4 nm apart. The high charge density of the aggrecan bottle-brush allows it to imbibe water, thereby maintaining tissue hydration and permeability, while also binding to cell-signaling molecules. In solution, aggrecan molecules respond differently to varying salt conditions, than other charged biological and synthetic polyelectrolytes like DNA and poly(acrylic acid) (Horkay, 2008). To probe the nature of its interactions with charged surfaces, we looked at the absorption patterns of aggrecan assemblies on controlled surfaces (polylysine, mica) under different ionic conditions, using Atomic Force Microscopy. We propose a simple model of the charge interactions, which relates the surface-adsorption patterns to the solution structures. The study may help understanding how aggrecan loss or degradation with age and joint disease affects tissue microstructure and physical properties.
Aggrecan is a negatively charged bottlebrush-shaped proteoglycan in the extracellular matrix, wit... more Aggrecan is a negatively charged bottlebrush-shaped proteoglycan in the extracellular matrix, with unique polyelectrolyte properties. Aggrecan-hyaluronic acid aggregates are responsible for the compressive resilience of articular cartilage. Unlike linear polyelectrolytes such as DNA, aggrecan is insensitive to the presence of multivalent counterions (e.g., calcium ions) and self-assembles into micro-gels in near-physiological salt solutions. These features are preserved by aggrecan adsorbed
ABSTRACTDNA is an anionic polyelectrolyte, which occupies a large volume in salt free solution du... more ABSTRACTDNA is an anionic polyelectrolyte, which occupies a large volume in salt free solution due to the coulomb repulsion between the charged groups. In the presence of high valence cations, DNA condenses into nanoparticles. DNA nanoparticles have generated a lot of interest as a preferred vehicle for delivering therapeutic DNA in gene therapy. The efficiency of gene delivery is determined by stability and compactness of the particles. However not much is known about the organization of DNA within the particles. The large polymer cations condense DNA rapidly, with no distinct intermediate stages that give insight into the arrangement of DNA within the nanoparticle. In our work, we form nanoparticles with short DNA strands to slow down the condensation process. The polymer cation is polyethyleneimine with grafted sugar moieties. Distinct intermediate stages are observed with Atomic Force Microscopy. The assembly occurs via the formation of fiber condensates, which appear to be the ...
ABSTRACTA quartz crystal microbalance (QCM) has been used to investigate polymer samples. The vap... more ABSTRACTA quartz crystal microbalance (QCM) has been used to investigate polymer samples. The vapor sorption of three different polymer samples (poly(vinyl acetate), polybutadiene and polydimethylsiloxane) was studied. The change in resonance frequency of the quartz sensor uniformly coated with polymer films was measured as a function of the film thickness and water absorption at different temperatures. The range of linear frequency vs. mass response was determined in the absence of absorbed water. The glass transition temperature of thin poly(vinyl acetate) films (10 nm <thickness< 1000 nm) was found to be in reasonable agreement with published values for macroscopic samples.
The effect of the simultaneous presence of monovalent and divalent cations on the thermodynamics ... more The effect of the simultaneous presence of monovalent and divalent cations on the thermodynamics of polyelectrolyte solutions is an incompletely solved problem. In physiological conditions, combinations of these ions affect structure formation in biopolymer systems. Dynamic light scattering measurements of the collective diffusion coefficient D and the osmotic compressibility of semidilute hyaluronan solutions containing different ratios of sodium and calcium ions are compared with simple polyelectrolyte models. Scaling relationships are proposed in terms of polymer concentration and ionic strength J of the added salt. Differences in the effects of sodium and calcium ions are found to be expressed only through J.
Nanomedicine: Nanotechnology, Biology and Medicine, 2012
Here we characterize the structure, stability and intracellular mode-of-action of DermaVir nanome... more Here we characterize the structure, stability and intracellular mode-of-action of DermaVir nanomedicine that is under clinical development for the treatment of HIV/AIDS. This nanomedicine is comprised of pathogen-like pDNA/PEIm nanoparticles (NPs) having the structure and function resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. Atomic force microscopy demonstrated spherical 100-200nm NPs with a smooth polymer surface protecting the pDNA in the core. Optical-absorption determined both the NP structural stability and biological activity relevant to their ability to escape from the endosome and release the pDNA at the nucleus. Salt, pH and temperature influence the nanomedicine shelf-life and intracellular stability. This approach facilitates the development of diverse polyplex nanomedicines where the delivered pDNA-expressed antigens induce immune responses to kill infected cells.
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Papers by Ferenc Horkay