Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Shells of compacted DNA as nanocontainers transporting proteins in multiplexed delivery.

Polyethyleneimine (PEI) polymers are known to compact DNA strands into spheroid, toroid, or rod structures. A formulation with mannose-grafted PEI (PEIm), however, was reported to compact DNA into ~100 nm spheroids that indented like thin-walled pressurized shells. The goal of the study is to understand why mannose bristles divert the traditional pathway of PEI-DNA compaction to produce shell-like structures, and to manipulate the process so that proteins can be packed into the core of the assembling shells for co-delivering DNA and proteins into cells. DLS, AFM, and TEM imaging provide a consistent picture that BSA proteins can be packed into the shells without altering the shell architecture, as long as the proteins were added during the time course of shell assembly. Force spectroscopy studies reveal that DNA shells that buckle also have a rich surface-coating of mannose, indicating that a micelle-like partitioning of hydrophobic and hydrophilic layers governs shell assembly. When HEK293T cells are spiked with BSA-laden DNA shells, co-transfection of DNA and BSA is observed at higher levels than control formulations. Distinct micron-sized features appear having both green fluorescence from BSA-FITC and blue fluorescence from NucBlue DNA stain, suggesting BSA release in nucleus and secretory granules. With DNA nanocontainers, proteins can take advantage of the efficiency of PEI-based DNA transfection for hitchhiking into cells while being shielded from the challenges of the intracellular route. DNA nanocontainers are rapid to assemble, not dependent on the DNA sequence, and can be adapted for different protein types; thereby having potential to serve as a high-throughput platform in scenarios where DNA and protein have to be released at the same site and time within cells (e.g., theranostics, multiplexed co-delivery, gene editing).

Establishing Rules for Self-Adhesion and Aggregation of N-Glycan Sugars Using Virus Glycan Shields.

The surfaces of cells and pathogens are covered with short polymers of sugars known as glycans. Complex N-glycans have a core of three mannose sugars with distal repeats of N-acetylglucosamine and galactose sugars terminating with sialic acid (SA). Long-range tough and short-range brittle self-adhesions were observed between SA and mannose residues, respectively, in ill-defined artificial monolayers. We investigated if and how these adhesions translate when the residues are presented in N-glycan architecture with SA at the surface and mannose at the core and with other glycan sugars. Two pseudotyped viruses with complex N-glycan shields were brought together in force spectroscopy (FS). At higher ramp rates, slime-like adhesions were observed between the shields, whereas Velcro-like adhesions were observed at lower rates. The higher approach rates compress the virus as a whole, and the self-adhesion between the surface SA is sampled. At the lower ramp rates, however, the complex glycan shield is penetrated and adhesion from the mannose core is accessed. The slime-like and Velcro-like adhesions were lost when SA and mannose were cleaved, respectively. While virus self-adhesion in forced contact was modulated by glycan penetrability, the self-aggregation of the freely diffusing virus was only determined by the surface sugar. Mannose-terminal viruses self-aggregated in solution, and SA-terminal ones required Ca2+ ions to self-aggregate. Viruses with galactose or N-acetylglucosamine surfaces did not self-aggregate, irrespective of whether or not a mannose core was present below the N-acetylglucosamine surface. Well-defined rules appear to govern the self-adhesion and -aggregation of N-glycosylated surfaces, regardless of whether the sugars are presented in an ill-defined monolayer, or N-glycan, or even polymer architecture.

Complex-type N-glycans on VSV-G pseudotyped HIV exhibit 'tough' sialic and 'brittle' mannose self-adhesions.

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.

Capturing 3D large-strain Euler-bending filament dynamics in fibrous media simulations; sample case of compression collapse in dendritic actin network.

Cytoskeletal networks to transmission towers are comprised of slender elements. Slender filaments bend and buckle more easily than stretch. Therefore a deforming network is expected to exhaust all possible bending-based modes before engaging filament stretch. While the large-strain bending critically determines fibrous-media response, simulations use small-strain and jointed approximations. At low resolution, these approximations inflate bending resistance and delay buckling onset. The proposed string-of-continuous-beams (SOCB) approach captures 3D nonlinear Euler bending of filaments with high fidelity at low cost. Bending geometry (i.e. angles and its differentials) is solved as primary variables, to fit a 5th order polynomial of the contour angle. Displacement, solved simultaneously as length conservation, is predicted with C3 and C6 smoothness between and within segments, using only 2 nodes. In the chosen analysis frame, in-plane and out-plane moments can be decoupled for arbitrarily-curved segments. Complex crosslink force-transfers can be specified. Simulations show that when a daughter branch is appended, the buckling resistance of a filament changes from linear to nonlinear before reversible collapse. An actin outcrop with 8 generations of mother-daughter branching produced the linear, nonlinear, and collapse regimes observed in compression experiments. 'Collapse' was a redistribution of outcrop forces following the buckling of few strands.

Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness.

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 resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.

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.

An engineering design approach to systems biology.

Measuring and modeling the integrated behavior of biomolecular-cellular networks is central to systems biology. Over several decades, systems biology has been shaped by quantitative biologists, physicists, mathematicians, and engineers in different ways. However, the basic and applied versions of systems biology are not typically distinguished, which blurs the separate aspirations of the field and its potential for real-world impact. Here, we articulate an engineering approach to systems biology, which applies educational philosophy, engineering design, and predictive models to solve contemporary problems in an age of biomedical Big Data. A concerted effort to train systems bioengineers will provide a versatile workforce capable of tackling the diverse challenges faced by the biotechnological and pharmaceutical sectors in a modern, information-dense economy.

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.

DNA nanoparticles with core-shell morphology.

Mannobiose-modified polyethylenimines (PEI) are used in gene therapy to generate nanoparticles of DNA that can be targeted to the antigen-presenting cells of the immune system. We report that the sugar modification alters the DNA organization within the nanoparticles from homogenous to shell-like packing. The depth-dependent packing of DNA within the nanoparticles was probed using AFM nano-indentation. Unmodified PEI-DNA nanoparticles display linear elastic properties and depth-independent mechanics, characteristic of homogenous materials. Mannobiose-modified nanoparticles, however, showed distinct force regimes that were dependent on indentation depth, with 'buckling'-like response that is reproducible and not due to particle failure. By comparison with theoretical studies of spherical shell mechanics, the structure of mannobiosylated particles was deduced to be a thin shell with wall thickness in the order of few nanometers, and a fluid-filled core. The shell-core structure is also consistent with observations of nanoparticle denting in altered solution conditions, with measurements of nanoparticle water content from AFM images, and with images of DNA distribution in Transmission Electron Microscopy.

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.

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

Structure and biological activity of pathogen-like synthetic nanomedicines.

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.

Averaged implicit hydrodynamic model of semiflexible filaments.

We introduce a method to incorporate hydrodynamic interaction in a model of semiflexible filament dynamics. Hydrodynamic screening and other hydrodynamic interaction effects lead to nonuniform drag along even a rigid filament, and cause bending fluctuations in semiflexible filaments, in addition to the nonuniform Brownian forces. We develop our hydrodynamics model from a string-of-beads idealization of filaments, and capture hydrodynamic interaction by Stokes superposition of the solvent flow around beads. However, instead of the commonly used first-order Stokes superposition, we do an equivalent of infinite-order superposition by solving for the true relative velocity or hydrodynamic velocity of the beads implicitly. We also avoid the computational cost of the string-of-beads idealization by assuming a single normal, parallel and angular hydrodynamic velocity over sections of beads, excluding the beads at the filament ends. We do not include the end beads in the averaging and solve for them separately instead, in order to better resolve the drag profiles along the filament. A large part of the hydrodynamic drag is typically concentrated at the filament ends. The averaged implicit hydrodynamics methods can be easily incorporated into a string-of-rods idealization of semiflexible filaments that was developed earlier by the authors. The earlier model was used to solve the Brownian dynamics of semiflexible filaments, but without hydrodynamic interactions incorporated. We validate our current model at each stage of development, and reproduce experimental observations on the mean-squared displacement of fluctuating actin filaments . We also show how hydrodynamic interaction confines a fluctuating actin filament between two stationary lateral filaments. Finally, preliminary examinations suggest that a large part of the observed velocity in the interior segments of a fluctuating filament can be attributed to induced solvent flow or hydrodynamic screening.

Probing Interactions between Aggrecan and Mica Surface by the Atomic Force Microscopy.

Aggrecan is a bottlebrush shaped macromolecule found in the extracellular matrix of cartilage. The negatively charged glycosaminoglycan (GAG) chains attached to its protein backbone give aggrecan molecules a high charge density, which is essential for exerting high osmotic swelling pressure and resisting compression under external load. In solution aggrecan assemblies are insensitive to the presence of calcium ions, and show distinct osmotic pressure versus concentration regimes. The aim of this study is to investigate the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well-controlled mica surfaces. The conformation of the aggrecan were visualized using Atomic Force Microscopy. On positively charged APS mica the GAG chains of the aggrecan molecules are distinguishable, and their average dimensions are practically unaffected by the presence of salt ions. With increasing aggrecan concentration they form clusters, and at higher concentrations they form a continuous monolayer of conforming molecules. On negatively charged mica, the extent of aggrecan adsorption varies with salt composition. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu.

Rods-on-string idealization captures semiflexible filament dynamics.

We present an approach to modeling the two-dimensional Brownian dynamics of semiflexible filaments in the worm-model description as uniform, isotropic, and continuously flexible. Experimental observations increasingly show that the mechanical behavior of semiflexible filament networks departs from conventional knowledge. A force-balance-based dynamic simulation of the filament networks has multiple advantages as an approach to understanding their anomalous mechanics. However, a major disadvantage is the difficulty of capturing filament hydrodynamics and bending mechanics in a computationally efficient and physically consistent manner. To that end, we propose a strategy for modeling semiflexible filaments which involves idealizing a semiflexible filament as a contiguous string of flexible rods, and considering the Brownian forces on it as Einsteinian-like point normal and tangential forces. By idealizing the filament as a string of rods, we avoid the complex hydrodynamic treatment involved in beads-on-string idealizations, and implement large-deflection beam mechanics and filament inextensibility in a natural manner, while reducing the computational size of the problem. By considering the Brownian forces as point normal and tangential forces, we decompose the Brownian forces on straight and curved segments into a combination of classical resultant forces and couples whose distribution is shown to be governed by the rod diffusion coefficients. The decomposition allows solution of the Euler beam equations to second-order continuity between segments and fifth-order continuity within segments. We show that the approach is physically consistent by capturing multiple Brownian phenomena ranging from the rigid to the semiflexible limit: the translational and rotational diffusion of rigid rods; the thermal fluctuation of semirigid cantilever filaments; and the shape, bending, and time relaxation of freely diffusing, semiflexible actin filaments.

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.

Deterministic material-based averaging theory model of collagen gel micromechanics.

Mechanics of collagen gels, like that of many tissues, is governed by events occurring on a length scale much smaller than the functional scale of the material. To deal with the challenge of incorporating deterministic micromechanics into a continuous macroscopic model, we have developed an averaging-theory-based modeling framework for collagen gels. The averaging volume, which is constructed around each integration point in a macroscopic finite-element model, is assumed to experience boundary deformations homogeneous with the macroscopic deformation field, and a micromechanical problem is solved to determine the average stress at the integration point. A two-dimensional version was implemented with the microstructure modeled as a network of nonlinear springs, and 500 segments were found to be sufficient to achieve statistical homogeneity. The method was then used to simulate the experiments of Tower et al. (Ann. Biomed. Eng., 30, pp. 1221-1233) who performed uniaxial extension of prealigned collagen gels. The simulation captured many qualitative features of the experiments, including a toe region and the realignment of the fibril network during extension. Finally, the method was applied to an idealized wound model based on the characterization measurements of Bowes et al. (Wound Repair Regen., 7, pp. 179-186). The model consisted of a strongly aligned "wound" region surrounded by a less strongly aligned "healthy" region. The alignment of the fibrils in the wound region led to reduced axial strains, and the alignment of the fibrils in the healthy region, combined with the greater effective stiffness of the wound region, caused rotation of the wound region during uniaxial stretch. Although the microscopic model in this study was relatively crude, the multiscale framework is general and could be employed in conjunction with any microstructural model.

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.