Volume Loss and Recovery in Bovine Knee Meniscus Loaded in Circumferential Tension.

Load-induced volume change is an important aspect of knee meniscus function because volume loss creates fluid pressure, which minimizes friction and helps support compressive loads. The knee meniscus is unusual amongst cartilaginous tissues in that it is loaded not only in axial compression, but also in circumferential tension between its tibial attachments. Despite the physiologic importance of the knee meniscus' tensile properties, its volumetric strain in tension has never been directly measured, and predictions of volume strain in the scientific literature are inconsistent. In this study, we apply uniaxial tension to bovine knee meniscus and use biplanar imaging to directly observe the resulting three-dimensional volume change and unloaded recovery, revealing that tension causes volumetric contraction. Compression is already known to also cause contraction; therefore, all major physiologic loads compress and pressurize the meniscus, inducing fluid outflow. Although passive unloaded recovery is often described as slow relative to loaded loss, here we show that at physiologic strains the volume recovery rate in the meniscus upon unloading is faster than the rate of volume loss. These measurements of volumetric strain are an important step toward a complete theory of knee meniscus fluid flow and load support.

Acute Repair of Meniscus Root Tear Partially Restores Joint Displacements as Measured With Magnetic Resonance Images and Loading in a Cadaveric Porcine Knee.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, increasing the risk of developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post-injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. The objective of this study was to quantify joint kinematics in an intact joint, after a meniscus root tear, and after suture repair in cadaveric porcine knees, a frequently used in vivo model. We utilized an magnetic resonance images-compatible loading device and novel use of a T1 vibe sequence to measure meniscus and femur displacements under physiological axial loads. We found that anterior root tear led to large meniscus displacements under physiological axial loading and that suture anchor repair reduced these displacements but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus pivoted in the axial plane. Methods from this study can be applied to assess altered joint kinematics following human knee injuries and evaluate repair strategies aimed to restore joint kinematics.

Overload in a Rat In Vivo Model of Synergist Ablation Induces Tendon Multiscale Structural and Functional Degeneration.

Tendon degeneration is typically described as an overuse injury with little distinction made between magnitude of load (overload) and number of cycles (overuse). Further, in vivo, animal models of tendon degeneration are mostly overuse models, where tendon damage is caused by a high number of load cycles. As a result, there is a lack of knowledge of how isolated overload leads to degeneration in tendons. A surgical model of synergist ablation (SynAb) overloads the target tendon, plantaris, by ablating its synergist tendon, Achilles. The objective of this study was to evaluate the structural and functional changes that occur following overload of plantaris tendon in a rat SynAb model. Tendon cross-sectional area (CSA) and shape changes were evaluated by longitudinal MR imaging up to 8 weeks postsurgery. Tissue-scale structural changes were evaluated by semiquantified histology and second harmonic generation microscopy. Fibril level changes were evaluated with serial block face scanning electron microscopy (SBF-SEM). Functional changes were evaluated using tension tests at the tissue and microscale using a custom testing system allowing both video and microscopy imaging. At 8 weeks, overloaded plantaris tendons exhibited degenerative changes including increases in CSA, cell density, collagen damage area fraction (DAF), and fibril diameter, and decreases in collagen alignment, modulus, and yield stress. To interpret the differences between overload and overuse in tendon, we introduce a new framework for tendon remodeling and degeneration that differentiates between the inputs of overload and overuse. In summary, isolated overload induces multiscale degenerative structural and functional changes in plantaris tendon.

Structure-Energetics-Property Relationships Support Computational Design of Photodegradable Pesticides.

Development of high-performing pesticides with tunable degradation properties is vital to increasing the safety and effectiveness of tomorrow's analogs. Chromophoric dissolved organic matter in the excited triple state (3CDOM*) is known to play a key role in the removal of pesticides via indirect photodegradation. However, the potential of these transformations to guide the design of safer chemicals has not yet been fully realized. Here, we report a two-tier computational framework developed to probe and predict both kinetics and thermodynamics of 3CDOM*-pesticide interactions. In the first tier, robust in silico models were constructed by fitting free energies obtained from density functional theory (DFT) calculations to cell potentials and second-order rate constants for the 3CDOM*-pesticide electron transfer. In the second tier, Gibbs free energies and corresponding free energy barriers, determined in solution using the Marcus theory, were applied to develop a quick yet accurate screening approach based on the frontier molecular orbital (FMO) Theory. Being highly mechanistic and spanning ca. 1500 unique 3CDOM*-pesticide interactions, our approach is both robust and broadly applicable. To that end, the outcomes of our computational models were integrated into an easy-to-use decision framework that can guide structure-based design of less persistent pesticide analogs.

Strain Distribution of Intact Rat Rotator Cuff Tendon-to-Bone Attachments and Attachments With Defects.

This study aimed to experimentally track the tissue-scale strains of the tendon-bone attachment with and without a localized defect. We hypothesized that attachments with a localized defect would develop strain concentrations and would be weaker than intact attachments. Uniaxial tensile tests and digital image correlation were performed on rat infraspinatus tendon-to-bone attachments with defects (defect group) and without defects (intact group). Biomechanical properties were calculated, and tissue-scale strain distributions were quantified for superior and inferior fibrous and calcified regions. At the macroscale, the defect group exhibited reduced stiffness (31.3±3.7 N/mm), reduced ultimate load (24.7±3.8 N), and reduced area under the curve at ultimate stress (3.7±1.5 J/m2) compared to intact attachments (42.4±4.3 N/mm, 39.3±3.7 N, and 5.6±1.4 J/m2, respectively). Transverse strain increased with increasing axial load in the fibrous region of the defect group but did not change for the intact group. Shear strain of the superior fibrous region was significantly higher in the defect group compared to intact group near yield load. This work experimentally identified that attachments may resist failure by distributing strain across the interface and that strain concentrations develop near attachment defects. By establishing the tissue-scale deformation patterns of the attachment, we gained insight into the micromechanical behavior of this interfacial tissue and bolstered our understanding of the deformation mechanisms associated with its ability to resist failure.

Advances in Quantification of Meniscus Tensile Mechanics Including Nonlinearity, Yield, and Failure.

The meniscus provides crucial knee function and damage to it leads to osteoarthritis of the articular cartilage. Accurate measurement of its mechanical properties is therefore important, but there is uncertainty about how the test procedure affects the results, and some key mechanical properties are reported using ad hoc criteria (modulus) or not reported at all (yield). This study quantifies the meniscus' stress-strain curve in circumferential and radial uniaxial tension. A fiber recruitment model was used to represent the toe region of the stress-strain curve, and new reproducible and objective procedures were implemented for identifying the yield point and measuring the elastic modulus. Patterns of strain heterogeneity were identified using strain field measurements. To resolve uncertainty regarding whether rupture location (i.e., midsubstance rupture versus at-grip rupture) influences the measured mechanical properties, types of rupture were classified in detail and compared. Dogbone (DB)-shaped specimens are often used to promote midsubstance rupture; to determine if this is effective, we compared DB and rectangle (R) specimens in both the radial and circumferential directions. In circumferential testing, we also compared expanded tab (ET) specimens under the hypothesis that this shape would more effectively secure the meniscus' curved fibers and thus produce a stiffer response. The fiber recruitment model produced excellent fits to the data. Full fiber recruitment occurred approximately at the yield point, strongly supporting the model's physical interpretation. The strain fields, especially shear and transverse strain, were extremely heterogeneous. The shear strain field was arranged in pronounced bands of alternating positive and negative strain in a pattern similar to the fascicle structure. The site and extent of failure showed great variation, but did not affect the measured mechanical properties. In circumferential tension, ET specimens underwent earlier and more rapid fiber recruitment, had less stretch at yield, and had greater elastic modulus and peak stress. No significant differences were observed between R and DB specimens in either circumferential or radial tension. Based on these results, ET specimens are recommended for circumferential tests and R specimens for radial tests. In addition to the data obtained, the procedural and modeling advances made in this study are a significant step forward for meniscus research and are applicable to other fibrous soft tissues.

Internal three-dimensional strains in human intervertebral discs under axial compression quantified noninvasively by magnetic resonance imaging and image registration.

Study objectives were to develop, validate, and apply a method to measure three-dimensional (3D) internal strains in intact human discs under axial compression. A custom-built loading device applied compression and permitted load-relaxation outside of the magnet while also maintaining compression and hydration during imaging. Strain was measured through registration of 300 µm isotropic resolution images. Excellent registration accuracy was achieved, with 94% and 65% overlap of disc volume and lamellae compared to manual segmentation, and an average Hausdorff, a measure of distance error, of 0.03 and 0.12 mm for disc volume and lamellae boundaries, respectively. Strain maps enabled qualitative visualization and quantitative regional annulus fibrosus (AF) strain analysis. Axial and circumferential strains were highest in the lateral AF and lowest in the anterior and posterior AF. Radial strains were lowest in the lateral AF, but highly variable. Overall, this study provided new methods that will be valuable in the design and evaluation surgical procedures and therapeutic interventions.

Acute repair of meniscus root tear partially restores joint displacements as measured with MRI and loading in a porcine knee.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, greatly increasing the risk for developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, this repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. However, many studies of meniscus function required opening the joint, which alters kinematics. The objective of this study was to use novel MRI methods to image the intact joint under axial load and measure the acute meniscus and femur displacements in an intact joint, after a meniscus root tear, and after suture repair in the porcine knee, a frequently used in vivo model. We found that anterior root tear led to large meniscus and femur displacements under physiological axial loading, and that suture anchor repair reduced these displacements, but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus rotated in the axial plane. This technique can be applied to evaluate the effect of knee injuries and to develop improved repair strategies to restore joint kinematics.

MRI-based measurement of in vivo disc mechanics in a young population due to flexion, extension, and diurnal loading.

Background: Intervertebral disc degeneration is often implicated in low back pain; however, discs with structural degeneration often do not cause pain. It may be that disc mechanics can provide better diagnosis and identification of the pain source. In cadaveric testing, the degenerated disc has altered mechanics, but in vivo, disc mechanics remain unknown. To measure in vivo disc mechanics, noninvasive methods must be developed to apply and measure physiological deformations. Aim: Thus, this study aimed to develop methods to measure disc mechanical function via noninvasive MRI during flexion and extension and after diurnal loading in a young population. This data will serve as baseline disc mechanics to later compare across ages and in patients. Materials & Methods: To accomplish this, subjects were imaged in the morning in a reference supine position, in flexion, in extension, and at the end of the day in a supine position. Disc deformations and vertebral motions were used to quantify disc axial strain, changes in wedge angle, and anterior-posterior (A-P) shear displacement. T2 weighted MRI was also used to evaluate disc degeneration via Pfirrmann grading and T2 time. All measures were then tested for effect of sex and disc level. Results: We found that flexion and extension caused level-dependent strains in the anterior and posterior of the disc, changes in wedge angle, and A-P shear displacements. Flexion had higher magnitude changes overall. Diurnal loading did not cause level-dependent strains but did cause small level-dependent changes in wedge angle and A-P shear displacements. Discussion: Correlations between disc degeneration and mechanics were largest in flexion, likely due to the smaller contribution of the facet joints in this condition. Conclusion: In summary, this study established methods to measure in vivo disc mechanical function via noninvasive MRI and established a baseline in a young population that may be compared to older subjects and clinical disorders in the future.

Biaxial tensile testing and constitutive modeling of human supraspinatus tendon.

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.

Structure-to-process design framework for developing safer pesticides.

Rational design of pesticides with tunable degradation properties and minimal ecotoxicity is among the grand challenges of green chemistry. While computational approaches have gained traction in predictive toxicology, current methods lack the necessary multifaceted approach and design-vectoring tools needed for system-based chemical development. Here, we report a tiered computational framework, which integrates kinetics and thermodynamics of indirect photodegradation with predictions of ecotoxicity and performance, based on cutoff values in mechanistically derived physicochemical properties and electronic parameters. Extensively validated against experimental data and applied to 700 pesticides on the U.S. Environmental Protection Agency's registry, our simple yet powerful approach can be used to screen existing molecules to identify application-ready candidates with desirable characteristics. By linking structural attributes to process-based outcomes and by quantifying trade-offs in safety, depletion, and performance, our method offers a user-friendly roadmap to rational design of novel pesticides.

Multiaxial validation of a finite element model of the intervertebral disc with multigenerational fibers to establish residual strain.

Finite element models of the intervertebral disc are used to address research questions that cannot be tested through typical experimentation. A disc model requires complex geometry and tissue properties to be accurately defined to mimic the physiological disc. The physiological disc possesses residual strain in the annulus fibrosus (AF) due to osmotic swelling and due to inherently pre-strained fibers. We developed a disc model with residual contributions due to swelling-only, and a multigeneration model with residual contributions due to both swelling and AF fiber pre-strain and validated it against organ-scale uniaxial, quasi-static and multiaxial, dynamic mechanical tests. In addition, we demonstrated the models' ability to mimic the opening angle observed following radial incision of bovine discs. Both models were validated against organ-scale experimental data. While the swelling only model responses were within the experimental 95% confidence interval, the multigeneration model offered outcomes closer to the experimental mean and had a bovine model opening angle within one SD of the experimental mean. The better outcomes for the multigeneration model, which allowed for the inclusion of inherently pre-strained fibers in AF, is likely due to its uniform fiber contribution throughout the AF. We conclude that the residual contribution of pre-strained fibers in the AF should be included to best simulate the physiological disc and its behaviors.

Impact of pulse sequence, analysis method, and signal to noise ratio on the accuracy of intervertebral disc T 2 measurement.

Noninvasive assessments of intervertebral disc health and degeneration are critical for addressing disc degeneration and low back pain. Magnetic resonance imaging (MRI) is exceptionally sensitive to tissue with high water content, and measurement of the MR transverse relaxation time, T 2, has been applied as a quantitative, continuous, and objective measure of disc degeneration that is linked to the water and matrix composition of the disc. However, T 2 measurement is susceptible to inaccuracies due to Rician noise, T 1 contamination, and stimulated echo effects. These error generators can all be controlled for with proper data collection and fitting methods. The objective of this study was to identify sequence parameters to appropriately acquire MR data and to establish curve fitting methods to accurately calculate disc T 2 in the presence of noise by correcting for Rician noise. To do so, we compared T 2 calculated from the typical monoexponential (MONO) fits and noise corrected exponential (NCEXP) fits. We examined how the selected sequence parameters altered the calculated T 2 in silico and in vivo. Typical MONO fits were frequently poor due to Rician noise, and NCEXP fits were more likely to provide accurate T 2 calculations. NCEXP is particularly less biased and less uncertain at low SNR. This study showed that the NCEXP using sequences with data from 20 echoes out to echo times of ~300 ms is the best method for calculating T 2 of discs. By acquiring signal data out to longer echo times and accounting for Rician noise, the curve fitting is more robust in calculating T 2 despite the noise in the data. This is particularly important when considering degenerate discs or AF tissue because the SNR of these regions is lower.

Structure, function, and defect tolerance with maturation of the radial tie fiber network in the knee meniscus.

The knee menisci are comprised of two orthogonal collagenous networks-circumferential and radial-that combine to enable efficient load bearing by the tissue in adults. Here, we assessed how the structural and functional characteristics of these networks developed over the course of skeletal maturation and determined the role of these fiber networks in defect tolerance with tissue injury. Imaging of the radial tie fiber (RTF) collagen structure in medial bovine menisci from fetal, juvenile, and adult specimens showed increasing heterogeneity, anisotropy, thickness, and density with skeletal development. The mechanical analysis showed that the tensile modulus in the radial direction did not change with skeletal development, though the resilience (in the radial direction) increased and the tolerance to defects in the circumferential direction decreased, in adult compared to fetal tissues. This loss of defect tolerance correlated with increased order in the RTF network in adult tissue. These data provide new insights into the role of the radial fiber network in meniscus function, will lead to improved clinical decision-making in the presence of a tear and may improve engineering efforts to reproduce this critical load-bearing structure in the knee.

Formation of filopodia-like bundles in vitro from a dendritic network.

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.

Helical fibrillar microstructure of tendon using serial block-face scanning electron microscopy and a mechanical model for interfibrillar load transfer.

Tendon's hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional (3D) microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer; however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy to investigate the 3D microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite-element modelling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

Off-axis response due to mechanical coupling across all six degrees of freedom in the human disc.

The kinematics of the intervertebral disc are defined by six degrees of freedom (DOF): three translations (Tz: axial compression, Tx: lateral shear, and Ty: anterior-posterior shear) and three rotations (Rz: torsion, Rx: flexion-extension, and Ry: lateral bending). There is some evidence that the six DOFs are mechanically coupled, such that loading in one DOF affects the mechanics of the other five "off-axis" DOFs, however, most studies have not controlled and/or measured all six DOFs simultaneously. Additionally, the relationships between disc geometry and disc mechanics are important for evaluation of data from different sized donor and patient discs. The objectives of this study were to quantify the mechanical behavior of the intervertebral disc in all six degrees of freedom (DOFs), measure the coupling between the applied motion in each DOF with the resulting off-axis motions, and test the hypothesis that disc geometry influences these mechanical behaviors. All off-axis displacements and rotations were significantly correlated with the applied DOF and were of similar magnitude as physiologically relevant motion, confirming that off-axis coupling is an important mechanical response. Interestingly, there were pairs of DOFs that were especially strongly coupled: lateral shear (Tx) and lateral bending (Ry), anterior-posterior shear (Ty) and flexion-extension (Rx), and compression (Tz) and torsion (Rz). Large off-axis shears may contribute to injury risk in bending and flexion. In addition, the disc responded to shear (Tx, Ty) and rotational loading (Rx, Ry, and Rz) by increasing in disc height in order to maintain the applied compressive load. Quantifying these mechanical behaviors across all six DOF are critical for designing and testing disc therapies, such as implants and tissue engineered constructs, and also for validating finite element models.

Short cracks in knee meniscus tissue cause strain concentrations, but do not reduce ultimate stress, in single-cycle uniaxial tension.

Tears are central to knee meniscus pathology and, from a mechanical perspective, are crack-like defects (cracks). In many materials, cracks create stress concentrations that cause progressive local rupture and reduce effective strength. It is currently unknown if cracks in meniscus have these consequences; if they do, this would have repercussions for management of meniscus pathology. The objective of this study was to determine if a short crack in meniscus tissue, which mimics a preclinical meniscus tear, (a) causes crack growth and reduces effective strength, (b) creates a near-tip strain concentration and (c) creates unloaded regions on either side of the crack. Specimens with and without cracks were tested in uniaxial tension and compared in terms of macroscopic stress-strain curves and digital image correlation strain fields. The strain fields were used as an indicator of stress concentrations and unloaded regions. Effective strength was found to be insensitive to the presence of a crack (potential effect < 0.86 s.d.; ß = 0.2), but significant strain concentrations, which have the potential to lead to long-term accumulation of tissue or cell damage, were observed near the crack tip.

In vitro assembly of filopodia-like bundles.

A breakthrough in understanding the mechanism of lamellipodial protrusion came from development of an in vitro model system, namely the rocketing movement of microbes and activated beads driven by actin comet tails (Cameron et al., 1999, 2000; Loisel et al., 1999; Theriot et al., 1994). As a model for investigation of the other major protrusive organelle, the filopodium, we developed in vitro systems for producing filopodia-like bundles (Vignjevic et al., 2003), one of which uses cytoplasmic extracts and another that reconstitutes like-like bundles from purified proteins. Beads coated with Arp2/3-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in star bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Similar to cells, the transition from a dendritic (lamellipodial) to a bundled (filopodial) organization is induced by depletion of capping protein, and add-back of this protein restores the dendritic mode. By use of purified proteins, a small number of components are sufficient for the assembly of filopodia-like bundles: WASP-coated beads, actin, Arp2/3 complex, and fascin. On the basis of analysis of this system, we proposed a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.

A comparison of stress in cracked fibrous tissue specimens with varied crack location, loading, and orientation using finite element analysis.

Cracks in fibrous soft tissue, such as intervertebral disc annulus fibrosus and knee meniscus, cause pain and compromise joint mechanics. A crack concentrates stress at its tip, making further failure and crack extension (fracture) more likely. Ex vivo mechanical testing is an important tool for studying the loading conditions required for crack extension, but prior work has shown that it is difficult to reproduce crack extension. Most prior work used edge crack specimens in uniaxial tension, with the crack 90° to the edge of the specimen. This configuration does not necessarily represent the loading conditions that cause in vivo crack extension. To find a potentially better choice for experiments aiming to reproduce crack extension, we used finite element analysis to compare, in factorial combination, (1) center crack vs. edge crack location, (2) biaxial vs. uniaxial loading, and (3) crack-fiber angles ranging from 0° to 90°. The simulated material was annulus fibrosus fibrocartilage with a single fiber family. We hypothesized that one of the simulated test cases would produce a stronger stress concentration than the commonly used uniaxially loaded 90° crack-fiber angle edge crack case. Stress concentrations were compared between cases in terms of fiber-parallel stress (representing risk of fiber rupture), fiber-perpendicular stress (representing risk of matrix rupture), and fiber shear stress (representing risk of fiber sliding). Fiber-perpendicular stress and fiber shear stress concentrations were greatest in edge crack specimens (of any crack-fiber angle) and center crack specimens with a 90° crack-fiber angle. However, unless the crack is parallel to the fiber direction, these stress components alone are insufficient to cause crack opening and extension. Fiber-parallel stress concentrations were greatest in center crack specimens with a 45° crack-fiber angle, either biaxially or uniaxially loaded. We therefore recommend that the 45° center crack case be tried in future experiments intended to study crack extension by fiber rupture.