Filament Stiffness Research Articles

Development of Flexible and Partly Water-Soluble Binder Systems for Metal Fused Filament Fabrication (MF3) of Ti-6Al-4V Parts.

Metal Fused Filament Fabrication provides a simple and cost-efficient way to produce dense metal parts with a homogenous microstructure. However, current limitations include the use of hazardous and expensive organic solvents during debinding for flexible filaments the stiffness of filaments made from partly water-soluble binder systems. In this study, the influence of various additives on different partly water-soluble binder systems, with regard to the flexibility and properties of the final parts, was investigated. Furthermore, a method using dynamic mechanical analysis to quantify the flexibility of filaments was introduced and successfully applied. For the first time, it was possible to produce flexible, partly water-soluble filaments with 60 vol.% solid content, which allowed the 3D printing of complex small and large parts with a high level of detail. After sintering, density values of up to 98.9% of theoretical density were achieved, which is significantly higher than those obtained with existing binder systems.

High-resolution yeast actin structures indicate the molecular mechanism of actin filament stiffening by cations

Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.

Switchable microscale stress response of actin-vimentin composites emerges from scale-dependent interactions.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and longtime stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Gaining new understanding of sarcomere length non-uniformities in skeletal muscles.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas (n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres.

Scale-dependent interactions enable emergent microrheological stress response of actin-vimentin composites.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and long-time stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

Engineering ssRNA tile filaments for (dis)assembly and membrane binding.

Cytoskeletal protein filaments such as actin and microtubules confer mechanical support to cells and facilitate many cellular functions such as motility and division. Recent years have witnessed the development of a variety of molecular scaffolds that mimic such filaments. Indeed, filaments that are programmable and compatible with biological systems may prove useful in studying or substituting such proteins. Here, we explore the use of ssRNA tiles to build and modify filaments in vitro. We engineer a number of functionalities that are crucial to the function of natural proteins filaments into the ssRNA tiles, including the abilities to assemble or disassemble filaments, to tune the filament stiffness, to induce membrane binding, and to bind proteins. This work paves the way for building dynamic cytoskeleton-mimicking systems made out of rationally designed ssRNA tiles that can be transcribed in natural or synthetic cells.

Correlating mechanical and rheological filament properties to processability and quality of 3D printed tablets using multiple linear regression

Filament formulation for FDM is a challenging and time-consuming process. Several pharmaceutical polymers are not feedable on their own. Due to inadequate filament formulation, 3D printed tablets can also exhibit poor uniformity of tablet attributes. To better understand filament formulation process, 23 filaments were prepared with the polymer mixing approach. To yield processable filaments, brittle and pliable polymers were combined. A 20 % addition of a pliable polymer to a brittle one resulted in filament processability and vice versa. Predictive statistical models for filament processability and uniformity of tablet attributes were established based on the mechanical and rheological properties of filaments. 15 input variables were correlated to 9 responses, which represent filament processability and tablet properties, by using multiple linear regression approach. Filament stiffness, assessed by indentation, and its square term were the only variables that determined the filament’s feedability. However, the resulting model is equipment-specific since different feeding mechanism exert different forces on the filaments. Additional models with good predictive power (R2pred > 0.50) were established for tablet width uniformity, drug release uniformity, tablet disintegration time uniformity and occurrence of disintegration, which are equipment-independent outputs. Therefore, the obtained model outcomes could be used in other research endeavours.

Palmitate-induced insulin resistance causes actin filament stiffness and GLUT4 mis-sorting without altered Akt signaling.

Skeletal muscle insulin resistance, a major contributor to Type 2 Diabetes, is linked to the consumption of saturated fats. This insulin resistance arises from failure of insulin-induced translocation of glucose transporter type 4 (GLUT4) to the plasma membrane to facilitate glucose uptake into muscle. The mechanisms of defective GLUT4 translocation are poorly understood, limiting development of insulin-sensitizing therapies targeting muscle glucose uptake. While many studies identify early insulin signaling defects and suggest they are responsible for insulin resistance, their cause-effect has been debated. Here, we find that the saturated fat palmitate (PA) causes insulin resistance of GLUT4 translocation in skeletal muscle myoblasts and myotubes without impairing signaling to Akt or AS160. Instead, PA altered two basal-state events: a) the intracellular localization of GLUT4 and its sorting towards a perinuclear storage compartment, and b) actin filament stiffness that prevents Rac1-dependent actin remodeling. These defects were triggered by distinct mechanisms, respectively protein palmitoylation and endoplasmic reticulum (ER) stress. Our findings highlight that saturated fats elicit muscle cell-autonomous dysregulation of the basal-state machinery required for GLUT4 translocation that 'primes' cells for insulin resistance.

Titin governs myocardial passive stiffness with major support from microtubules and actin and the extracellular matrix.

Myocardial passive stiffness is crucial for the heart's pump function and is determined by mechanical elements, including the extracellular matrix and cytoskeletal filaments; however, their individual contributions are controversially discussed and difficult to quantify. In this study, we targeted the cytoskeletal filaments in a mouse model, which enables the specific, acute and complete cleavage of the sarcomeric titin springs. We show in vitro that each cytoskeletal filament's stiffness contribution varies depending on whether the elastic or the viscous forces are considered and on strain level. Titin governs myocardial elastic forces, with the largest contribution provided at both low and high strain. Viscous force contributions are more uniformly distributed among the microtubules, titin and actin. The extracellular matrix contributes at high strain. The remaining forces after total target element disruption are likely derived from desmin filaments. Our findings answer longstanding questions about cardiac mechanical architecture and allow better targeting of passive myocardial stiffness in heart failure.

Nonlinear elastic behaviors and deformation mechanisms of nano-structured crosslinked biopolymer networks

Cells always undergo large deformation in response to external stimulations or internal stresses in many cell functions (e.g., cell migration, cell division and cell growth). When undergoing large deformation, crosslinked actin filament networks (CAFNs) always show strong nonlinear elasticity to maintain the cell shape and integrity, known as strain stiffening, which plays a crucial role in many cell functions. To investigate the nonlinear elastic behaviors of CAFNs, a three-dimensional representative volume element model is used to perform finite element method simulations. Simulation results show that actin filament volume fraction, crosslinking density and components’ Young’s moduli show significant influences on the nonlinear elastic behaviors of CAFNs. In addition, the shear stress–strain curves of CAFNs highly depend on the bending stiffness and tensile stiffness of filamins as well as the bending stiffness of actin filaments, however, they are almost insensitive to the tensile stiffness of actin filaments. The present work not only sheds light on the nonlinear elastic behaviors of CAFNs but also provides a valuable reference for developing advanced artificial composite structures that can be used as semi-flexible biomedical scaffolds and wearable electronics.

Nonlinear Microscale Mechanics of Actin Networks Governed by Coupling of Filament Crosslinking and Stabilization.

Actin plays a vital role in maintaining the stability and rigidity of biological cells while allowing for cell motility and shape change. The semiflexible nature of actin filaments-along with the myriad actin-binding proteins (ABPs) that serve to crosslink, bundle, and stabilize filaments-are central to this multifunctionality. The effect of ABPs on the structural and mechanical properties of actin networks has been the topic of fervent investigation over the past few decades. Yet, the combined impact of filament stabilization, stiffening and crosslinking via ABPs on the mechanical response of actin networks has yet to be explored. Here, we perform optical tweezers microrheology measurements to characterize the nonlinear force response and relaxation dynamics of actin networks in the presence of varying concentrations of α-actinin, which transiently crosslinks actin filaments, and phalloidin, which stabilizes filamentous actin and increases its persistence length. We show that crosslinking and stabilization can act both synergistically and antagonistically to tune the network resistance to nonlinear straining. For example, phalloidin stabilization leads to enhanced elastic response and reduced dissipation at large strains and timescales, while the initial microscale force response is reduced compared to networks without phalloidin. Moreover, we find that stabilization switches this initial response from that of stress stiffening to softening despite the increased filament stiffness that phalloidin confers. Finally, we show that both crosslinking and stabilization are necessary to elicit these emergent features, while the effect of stabilization on networks without crosslinkers is much more subdued. We suggest that these intriguing mechanical properties arise from the competition and cooperation between filament connectivity, bundling, and rigidification, shedding light on how ABPs with distinct roles can act in concert to mediate diverse mechanical properties of the cytoskeleton and bio-inspired polymeric materials.

FP.02 Mutation in KBTBD13 causes stiffening of thin filaments in skeletal muscle

Nemaline myopathy is caused by mutations in genes encoding thin filament proteins. Nemaline myopathy-type 6 (NEM6) is caused by mutations in <i>KBTBD13</i>. NEM6 patients display muscle weakness and slow kinetics of muscle relaxation. Whether KBTBD13 plays a role in thin filament structure/function, and whether such role can explain the NEM6 phenotype, is incompletely understood. Therefore, we developed a KBTBD13R408C–knockin (KI) mouse model. In line with the patients' phenotype, KI mice exhibit nemaline rods and slow relaxation kinetics. We used low-angle x-ray diffraction to study whether structural changes in the myofilaments can explain the contractile phenotype of KI mice. Intact soleus and extensor digitorum longus muscles (EDL) were dissected from wt and KI mice. Low angle x-ray diffraction experiments were conducted at the BioCAT beamline, Argonne National Laboratories. Muscles were mounted between a force transducer and a fixed hook, exposed to the x-ray beam during relaxed and maximal tetanic conditions. Images were recorded with 15ms acquisition time using a Pilatus 3xM1 detector set at 1.26 meter distance from the muscle. Results show that in relaxed soleus muscles the actin monomer spacing (27A) and myosin backbone spacing (28A) are shorter in KI compared to wt mice (2.7329 vs 2.7343nm; 2.8764nm vs 2.8797nm). Upon maximal tetanic activation, which was comparable between KI and wt muscles, the 27A spacing increased less in KI compared to wt muscles, indicating increased stiffness. Upon maximal tetanic activation, the 28A spacing increased in wt muscles, but this increase was comparable to that in KI mice. EDL muscle of KI mice showed no differences in actin monomer or myosin backbone spacings. Results indicate that thin filament stiffness, but not thick filament stiffness, is increased in KI mice, and that this increase is muscle-type specific. Sarcomere modeling shows that the increased stiffness of thin filaments contributes to slow relaxation kinetics.

Measurement of the persistence length of cytoskeletal filaments using curvature distributions

Cytoskeletal filaments, such as microtubules and actin filaments, play important roles in the mechanical integrity of cells and the ability of cells to respond to their environment. Measuring the mechanical properties of cytoskeletal structures is crucial for gaining insight into intracellular mechanical stresses and their role in regulating cellular processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring filament deformations, such as Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature distributions can be used as an alternative tool to quantify biofilament deformations, and investigate how the apparent stiffness of filaments depends on the resolution and noise of the imaging system. We present analytical calculations of the scaling curvature distributions as a function of filament discretization, and test our predictions by comparing Monte Carlo simulations with results from existing techniques. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of nonfunctional motors, and calculate the persistence length of these filaments. The presented curvature analysis is significantly more accurate compared with existing approaches for small data sets, and can be readily applied to both in vitro and in vivo filament data through the use of the open-source codes we provide.

Effect of Base Oil and Thickener on Texture and Flow of Lubricating Greases: Insights from Bulk Rheometry, Optical Microrheology and Electron Microscopy

The structure and flow behavior of lubricating greases depend on the base oil and the type and concentration of the dissolved thickener. In this study, the linear viscoelastic properties of greases were characterized by combining oscillatory shear and squeeze flow covering a broad frequency range (0.1–105 rad s−1). Multiple-particle tracking (MPT) microrheology and scanning electron microscopy (SEM) provided further insight into local viscoelastic properties and sample structure on a submicron-length scale. The type and viscosity of the base oil did not affect the absolute value of the complex viscosity and the filament shape formed by a given thickener. High-frequency shear modulus data, however, indicated that the thickener lithium 12-hydroxystearate formed stiffer networks/filaments in poly-α-olefins than in mineral oils. As expected, the viscosity increased with increased thickener concentrations, but microscopy and high-frequency rheometry revealed that the thickness, length, and stiffness of the individual filaments did not change. In mineral oil, the 12-hydroxystearate thickeners yielded higher viscosity than the corresponding stearates with the same metal ion. The filamentous lithium thickeners created stronger networks than the roundish aggregates formed by magnesium and zinc stearate. Network mesh sizes varying between approximately 100 nm and 300 nm were consistently determined from SEM image analysis and MPT experiments. The MPT experiments further disclosed the existence of gel-like precursors of approximately 130 µm at thickener concentrations far below the critical value at which a sample-spanning network resulting in a characteristic grease texture is formed.

Flow past a rotationally oscillating cylinder with an attached flexible filament

Experimental studies are conducted on a rotationally oscillating cylinder with an attached flexible filament at a Reynolds number of 150. Parametric studies are carried out to investigate the effect of cylinder forcing parameters and filament stiffness on the resultant wake structure. The diagnostics are flow visualization using the laser-induced fluorescence technique, frequency measurement using a hot film, and characterization of the velocity and vorticity field using planar particle image velocimetry. The streamwise force and power are estimated through control volume analysis, using a modified formulation, which considers the streamwise and transverse velocity fluctuations in the wake. These terms become important in a flow field where asymmetric wakes are observed. An attached filament significantly modifies the flow past a rotationally oscillating cylinder from a Bénard–Kármán vortex street to a reverse Bénard–Kármán vortex street, albeit over a certain range of Strouhal number, $St_{A} \sim 0.25\text {--}0.5$ , encountered in nature in flapping flight/fish locomotion and in the flow past pitching airfoils. The transition from a Kármán vortex street to a reverse Kármán vortex street precedes the drag-to-thrust transition. The mechanism of unsteady thrust generation is discussed. Maximum thrust is generated at the instants when vortices are shed in the wake from the filament tip. At $St_{A} &gt; 0.4$ , a deflected wake associated with the shedding of an asymmetric vortex street is observed. Filament flexibility delays the formation of an asymmetric wake. Wake symmetry is governed by the time instant at which a vortex pair is shed in the wake from the filament tip.

Mechanical characterization of hydrolysis effects on the stiffness of bioabsorbable polymeric filaments: An experimental and modeling approach based on a simple constitutive damage model

This manuscript presents an experimental and modeling approach in order to characterize the stiffness loss of bioabsorbable polymer filaments due to hydrolysis. In this regard, bioabsorbable suture yarns (poly(lactic-co-glycolic) acid—PLGA) were chosen as a representative material for the present investigation. The observed mechanical response was characterized by means of a thermodynamically consistent constitutive variational framework. Usually, two different damage variables are assumed to take place in this class of materials: a hydrolytic damage (long-term degradation) and a strain-driven damage (short-term degradation). This work concerns the proposition of a constitutive model that only considers the hydrolytic damage, in which a specific strain energy and a proper dissipation damage potential were tailored to model the tested material. A nonlinear curve fitting procedure based on Particle Swarm Optimization was performed to identify the constitutive parameters. A set of numerical simulations demonstrates the effectiveness of the proposed constitutive model to predict damage-induced creep and damage-induced stress relaxation, behaviors that can be used as design criteria in absorbable implants. The main achieved results show that the proposed constitutive approach leads to a simple but effective model capable to drive the first steps in the design of absorbable biomedical devices. The present variational framework can be extended to study the constitutive response of other bioabsorbable polymers, accounting for viscous and/or plastic behaviors.

Role of release modifiers to modulate drug release from fused deposition modelling (FDM) 3D printed tablets

Although hot melt extrusion (HME) has been used in combination with fused deposition modelling (FDM) three-dimensional printing (3DP), suitable feedstock materials such as polymeric filaments with optimum properties are still limited. In this study, various release modifying excipients, namely, poly(vinyl alcohol) (PVA), Soluplus®, polyethylene glycol (PEG) 6000, Eudragit® RL PO/RS PO, hydroxypropyl methylcellulose (HPMC) K4M/E10M/K100M, Kollidon® vinyl acetate 64 (VA 64)/17PF/30, were used as a release modulating tool to control the drug release from 3D printed sustained release tablets. Ibuprofen (as the model drug) and ethyl cellulose (as the polymeric matrix), along with various release modifiers, were blended and extruded into filaments through a twin-screw extruder. Then these filaments were printed into cylindrical tablets through FDM 3DP technique and their surface morphology, thermal stability, solid-state, mechanical properties, dose accuracy and drug release behaviors were investigated. The solid-state analysis of 3D printed tablets exhibited the amorphous nature of the drug dispersed in the polymer matrices. Although all these prepared filaments could be successfully printed without failing during the FDM 3DP process, the mechanical characterization showed that the filament stiffness and brittleness could be adjusted significantly by changing the type of release modifiers. Moreover, in vitro drug release studies revealed that the drug release could simply be controlled over 24 h by only changing the type of release modifiers. All ibuprofen (IBP) loaded 3D printed tablets with ethyl cellulose (EC) matrix, especially with PEG as the release modifier, showed great potential in releasing IBP in a zero-order reaction. In conclusion, all the results illustrated that the HME/FDM approach and optimized formulation compositions can be an attractive option for the development of pharmaceutical tablets and implants where adjustable drug release patterns are necessary.

Investigation of Interlayer Interface Strength and Print Morphology Effects in Fused Deposition Modeling 3D-Printed PLA.

Fused deposition modeling polymer 3D printing has become a popular versatile additive manufacturing technology. However, there are limitations to the mechanical properties due to the layer-by-layer deposition approach. The relatively low strength of the interface between layers is the cause for potential microstructural weak points in such printed components. The interface strength of 3D-printed Polylactic Acid (PLA) polymer was determined through physical tensile testing in combination with microstructural finite element method (FEM) simulations. A custom tensile specimen was created to isolate the interlayer interfaces for direct testing of interface strength. Tensile tests resulted in an average 2.4 GPa stiffness and an average 22.8 MPa tensile strength for printed specimens, corresponding to a 32.4% and 47.8% reduction from the bulk filament stiffness and strength, respectively. Sectioned tensile specimens were observed under a digital microscope to examine microstructural features such as inter-layer gaps, extrusion cross-section, and voids. These were measured to create accurate FEM microstructural model geometries. The brittle fracture that occurred during the tensile testing was due to debonding of the interfaces. This was represented in Abaqus by using cohesive surfaces. Interface strength was inferred by varying the strength of the cohesive surfaces until the simulation mechanical response matched the physical tests. The resulting interface strength of the PLA polymer was 33.75 MPa on average, corresponding to a 22.5% reduction from bulk properties. Potential improvements to the overall strength of the 3D printed PLA were investigated in simulation by parameterizing improved gap morphologies. As the size of the interlayer gaps decreased, the stiffness and strength of the printed parts improved, whereas completely eliminating gaps resulted in a potential 16.1% improvement in material stiffness and 19.8% improvement in strength. These models show that significant improvements can be made to the overall printed part performance by optimizing the printing process and eliminating inner voids.

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.