Preethi L. Chandran

Affine versus non-affine fibril kinematics in collagen networks : Theoretical studies of network behavior

The microstructure of tissues and tissue equivalents (TEs) plays a critical role in determining the mechanical properties thereof. One of the key challenges in constitutive modeling of TEs is incorporating the kinematics at both the macroscopic and the microscopic scale. Models of fibrous microstructure commonly assume fibrils to move homogeneously, that is affine with the macroscopic deformation. While intuitive for situations of fibril-matrix load transfer, the relevance of the affine assumption is less clear when primary load transfer is from fibril to fibril. The microstructure of TEs is a hydrated network of collagen fibrils, making its microstructural kinematics an open question. Numerical simulation of uniaxial extensile behavior in planar TE networks was performed with fibril kinematics dictated by the network model and by the affine model. The average fibril orientation evolved similarly with strain for both models. The individual fibril kinematics, however, were markedly different. There was no correlation between fibril strain and orientation in the network model, and fibril strains were contained by extensive reorientation. As a result, the macroscopic stress given by the network model was roughly threefold lower than the affine model. Also, the network model showed a toe region, where fibril reorientation precluded the development of significant fibril strain. We conclude that network fibril kinematics are not governed by affine principles, an important consideration in the understanding of tissue and TE mechanics, especially when load bearing is primarily by an interconnected fibril network.

Microstructural mechanics of Collagen gels in confined compression: Poroelasticity, viscoelasticity, and collapse

BACKGROUND Collagen gels are important as platforms for in vitro study of cell behavior and as prototypical bioartificial tissues, but their mechanical behavior, particularly on the microscopic scale, is still poorly understood. METHOD OF APPROACH Collagen gels were studied in step (10% strain in 0.05 s) and ramp (0.1%/s strain rate for 100 s) confined compression. Real-time birefringence mapping gave the local collagen concentration and orientation along with piston stress. Variations in the retardation allowed material-point tracking and qualitative determination of the strain distribution. RESULTS Ramp tests showed classical poroelastic behavior: compression near the piston and relaxation to a uniform state. Step tests, however, showed an irreversibly collapsed region near the piston. CONCLUSIONS Our results suggest that interstitial flow and fibril bending at crosslinks are the dominant mechanical processes during compression, and that fibril bending is reversible before collapse.

Collagen Fiber Alignment Does Not Explain Mechanical Anisotropy in Fibroblast Populated Collagen Gels

Many load-bearing soft tissues exhibit mechanical anisotropy. In order to understand the behavior of natural tissues and to create tissue engineered replacements, quantitative relationships must be developed between the tissue structures and their mechanical behavior. We used a novel collagen gel system to test the hypothesis that collagen fiber alignment is the primary mechanism for the mechanical anisotropy we have reported in structurally anisotropic gels. Loading constraints applied during culture were used to control the structural organization of the collagen fibers of fibroblast populated collagen gels. Gels constrained uniaxially during culture developed fiber alignment and a high degree of mechanical anisotropy, while gels constrained biaxially remained isotropic with randomly distributed collagen fibers. We hypothesized that the mechanical anisotropy that developed in these gels was due primarily to collagen fiber orientation. We tested this hypothesis using two mathematical models that incorporated measured collagen fiber orientations: a structural continuum model that assumes affine fiber kinematics and a network model that allows for nonaffine fiber kinematics. Collagen fiber mechanical properties were determined by fitting biaxial mechanical test data from isotropic collagen gels. The fiber properties of each isotropic gel were then used to predict the biaxial mechanical behavior of paired anisotropic gels. Both models accurately described the isotropic collagen gel behavior. However, the structural continuum model dramatically underestimated the level of mechanical anisotropy in aligned collagen gels despite incorporation of measured fiber orientations; when estimated remodeling-induced changes in collagen fiber length were included, the continuum model slightly overestimated mechanical anisotropy. The network model provided the closest match to experimental data from aligned collagen gels, but still did not fully explain the observed mechanics. Two different modeling approaches showed that the level of collagen fiber alignment in our uniaxially constrained gels cannot explain the high degree of mechanical anisotropy observed in these gels. Our modeling results suggest that remodeling-induced redistribution of collagen fiber lengths, nonaffine fiber kinematics, or some combination of these effects must also be considered in order to explain the dramatic mechanical anisotropy observed in this collagen gel model system.

Aggrecan, an unusual polyelectrolyte: review of solution behavior and physiological implications.

Aggrecan is a high-molecular-weight, bottlebrush-shaped, negatively charged biopolymer that forms supermolecular complexes with hyaluronic acid. In the extracellular matrix of cartilage, aggrecan-hyaluronic acid complexes are interspersed in a collagen meshwork and provide the osmotic properties required to resist deswelling under compressive load. In this review we compile aggrecan solution behavior from different experimental techniques, and discuss them in the context of concentration regimes that were identified in osmotic pressure experiments. At low concentrations, aggrecan exhibits microgel-like behavior. With increasing concentration, the bottlebrushes self-assemble into large complexes. In the physiological concentration range (2<c(aggrecan)<8% w/w), the physical properties of the solution are dominated by repulsive electrostatic interactions between aggrecan complexes. We discuss the consequences of the bottlebrush architecture on the polyelectrolyte characteristics of the aggrecan molecule, and its implications for cartilage properties and function.

Structure and biological activity of pathogen-like synthetic nanomedicines

UNLABELLED 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 comprises 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 - 200 nm 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 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. FROM THE CLINICAL EDITOR The authors investigated DermaVir nanomedicine comprised of pathogen-like pDNA/PEIm nanoparticles with structure and function resembling spherical viruses. DermaVir delivery of pDNA expresses antigens that induce immune responses to kill HIV infected cells.

Imaging Self-Assembly Dependent Spatial Distribution of Small Molecules in a Cellular Environment

Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in a cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit a different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in a cellular environment. Moreover, cell viability tests suggest that the states and the locations of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work demonstrates self-assembly as a key factor for dictating the spatial distribution of small molecules in a cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in a cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells.

Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking.

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.

Unusual Salt and pH Induced Changes in Polyethylenimine Solutions.

Linear PEI is a cationic polymer commonly used for complexing DNA into nanoparticles for cell-transfection and gene-therapy applications. The polymer has closely-spaced amines with weak-base protonation capacity, and a hydrophobic backbone that is kept unaggregated by intra-chain repulsion. As a result, in solution PEI exhibits multiple buffering mechanisms, and polyelectrolyte states that shift between aggregated and free forms. We studied the interplay between the aggregation and protonation behavior of 2.5 kDa linear PEI by pH probing, vapor pressure osmometry, dynamic light scattering, and ninhydrin assay. Our results indicate that: At neutral pH, the PEI chains are associated and the addition of NaCl initially reduces and then increases the extent of association. The aggregate form is uncollapsed and co-exists with the free chains. PEI buffering occurs due to continuous or discontinuous charging between stalled states. Ninhydrin assay tracks the number of unprotonated amines in PEI. The size of PEI-DNA complexes is not significantly affected by the free vs. aggregated state of the PEI polymer. Despite its simple chemical structure, linear PEI displays intricate solution dynamics, which can be harnessed for environment-sensitive biomaterials and for overcoming current challenges with DNA delivery.

Microstructure-Based, Multiscale Modeling for the Mechanical Behavior of Hydrated Fiber Networks

A multiscale formulation is derived for the mechanics of a dilute fiber network microstructure, as occurs in in vitro reconstituted collagen gels, to accommodate the deterministic solution of a uniform-stress condition in the fiber network. The macroscale two-phase equations are derived based on the integral volume-averaging approach of the spatial averaging theorem, modified for the averaging volume to deform materially in the solid phase and thereby ensuring consistent network mass conservation. For low-Reynolds-number fiber-fluid interaction with no hydrodynamic interaction between fibers, the macroscale Darcy law arises naturally as a function of average fiber orientation and volume fraction, with no additional empirical specification. The macroscale equations are solved using finite element analysis with the averaging volumes centered at Gauss points of integration. The macroscale solid stress and fluid velocity are obtained by microscale deterministic solution of network and Stokesian mechanics with...

Mannose Surfaces Exhibit Self-Latching, Water Structuring, and Resilience to Chaotropes: Implications for Pathogen Virulence

Several viral and fungal pathogens, including HIV, SARS, Dengue, Ebola, and Cryptococcus neoformans, display a preponderance of mannose residues on their surface, particularly during the infection cycle or in harsh environments. The innate immune system, on the other hand, abounds in mannose receptors which recognize mannose residues on pathogens and trigger their phagocytosis. We pose the question if there is an advantage for pathogens to display mannose on their surface, despite these residues being recognized by the immune system. The surface properties and interactions of opposing monolayers of mannobiose (disaccharide of mannose) were probed using atomic force spectroscopy. Unlike its diastereoisomer lactose, mannobiose molecules exhibited lateral packing interactions that manifest on the surface scale as a self-recognizing latch. A break-in force is required for opposing surfaces to penetrate and a breakout (or self-adhesion force) of similar magnitude is required for penetrated surfaces to separate. A hierarchy of self-adhesion forces was distinguished as occurring at the single residue (∼25 pN), cluster (∼250 pN), monolayer (∼1.1 nN), and supramonolayer level. The break-in force and break-out force appear resilient to the presence of simple chaotropes that attenuate a layer of structured water around the mannose surface. The layer of structured water otherwise extends to distances several times longer than a mannobiose residue, indicating a long-range propagation of the hydrogen bonding imposed by the residues. The span of the structured water increases with the velocity of an approaching surface, similar to shear thickening, but fissures at higher approach velocities. Our studies suggest that mannose residues could guide interpathogen interactions, such as in biofilms, and serve as a moated fortress for pathogens to hide behind to resist detection and harsh environments.