Effects of extracellular matrix viscoelasticity on cellular behaviour.

Substantial research over the past two decades has established that extracellular matrix (ECM) elasticity, or stiffness, affects fundamental cellular processes, including spreading, growth, proliferation, migration, differentiation and organoid formation. Linearly elastic polyacrylamide hydrogels and polydimethylsiloxane (PDMS) elastomers coated with ECM proteins are widely used to assess the role of stiffness, and results from such experiments are often assumed to reproduce the effect of the mechanical environment experienced by cells in vivo. However, tissues and ECMs are not linearly elastic materials-they exhibit far more complex mechanical behaviours, including viscoelasticity (a time-dependent response to loading or deformation), as well as mechanical plasticity and nonlinear elasticity. Here we review the complex mechanical behaviours of tissues and ECMs, discuss the effect of ECM viscoelasticity on cells, and describe the potential use of viscoelastic biomaterials in regenerative medicine. Recent work has revealed that matrix viscoelasticity regulates these same fundamental cell processes, and can promote behaviours that are not observed with elastic hydrogels in both two- and three-dimensional culture microenvironments. These findings have provided insights into cell-matrix interactions and how these interactions differentially modulate mechano-sensitive molecular pathways in cells. Moreover, these results suggest design guidelines for the next generation of biomaterials, with the goal of matching tissue and ECM mechanics for in vitro tissue models and applications in regenerative medicine.

Emergence of tissue-like mechanics from fibrous networks confined by close-packed cells.

The viscoelasticity of the crosslinked semiflexible polymer networks-such as the internal cytoskeleton and the extracellular matrix-that provide shape and mechanical resistance against deformation is assumed to dominate tissue mechanics. However, the mechanical responses of soft tissues and semiflexible polymer gels differ in many respects. Tissues stiffen in compression but not in extension1-5, whereas semiflexible polymer networks soften in compression and stiffen in extension6,7. In shear deformation, semiflexible polymer gels stiffen with increasing strain, but tissues do not1-8. Here we use multiple experimental systems and a theoretical model to show that a combination of nonlinear polymer network elasticity and particle (cell) inclusions is essential to mimic tissue mechanics that cannot be reproduced by either biopolymer networks or colloidal particle systems alone. Tissue rheology emerges from an interplay between strain-stiffening polymer networks and volume-conserving cells within them. Polymer networks that soften in compression but stiffen in extension can be converted to materials that stiffen in compression but not in extension by including within the network either cells or inert particles to restrict the relaxation modes of the fibrous networks that surround them. Particle inclusions also suppress stiffening in shear deformation; when the particle volume fraction is low, they have little effect on the elasticity of the polymer networks. However, as the particles become more closely packed, the material switches from compression softening to compression stiffening. The emergence of an elastic response in these composite materials has implications for how tissue stiffness is altered in disease and can lead to cellular dysfunction9-11. Additionally, the findings could be used in the design of biomaterials with physiologically relevant mechanical properties.

Compression stiffening of fibrous networks with stiff inclusions.

Tissues commonly consist of cells embedded within a fibrous biopolymer network. Whereas cell-free reconstituted biopolymer networks typically soften under applied uniaxial compression, various tissues, including liver, brain, and fat, have been observed to instead stiffen when compressed. The mechanism for this compression-stiffening effect is not yet clear. Here, we demonstrate that when a material composed of stiff inclusions embedded in a fibrous network is compressed, heterogeneous rearrangement of the inclusions can induce tension within the interstitial network, leading to a macroscopic crossover from an initial bending-dominated softening regime to a stretching-dominated stiffening regime, which occurs before and independently of jamming of the inclusions. Using a coarse-grained particle-network model, we first establish a phase diagram for compression-driven, stretching-dominated stress propagation and jamming in uniaxially compressed two- and three-dimensional systems. Then, we demonstrate that a more detailed computational model of stiff inclusions in a subisostatic semiflexible fiber network exhibits quantitative agreement with the predictions of our coarse-grained model as well as qualitative agreement with experiments.

The vimentin cytoskeleton: when polymer physics meets cell biology.

The proper functions of tissues depend on the ability of cells to withstand stress and maintain shape. Central to this process is the cytoskeleton, comprised of three polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). IF proteins are among the most abundant cytoskeletal proteins in cells; yet they remain some of the least understood. Their structure and function deviate from those of their cytoskeletal partners, F-actin and microtubules. IF networks show a unique combination of extensibility, flexibility and toughness that confers mechanical resilience to the cell. Vimentin is an IF protein expressed in mesenchymal cells. This review highlights exciting new results on the physical biology of vimentin intermediate filaments and their role in allowing whole cells and tissues to cope with stress.

Salmon fibrinogen and chitosan scaffold for tissue engineering: in vitro and in vivo evaluation.

3D fibrous scaffolds have received much recent attention in regenerative medicine. Use of fibrous scaffolds has shown promising results in tissue engineering and wound healing. Here we report the development and properties of a novel fibrous scaffold that is useful for promoting wound healing. A scaffold made of salmon fibrinogen and chitosan is produced by electrospinning, resulting in a biocompatible material mimicking the structure of the native extracellular matrix (ECM) with suitable biochemical and mechanical properties. The scaffold is produced without the need for enzymes, in particular thrombin, but is fully compatible with their addition if needed. Human dermal fibroblasts cultured on this scaffold showed progressive proliferation for 14 days. Split-thickness experimental skin wounds treated and untreated were compared in a 10-day follow-up period. Wound healing was more effective using the fibrinogen-chitosan scaffold than in untreated wounds. This scaffold could be applicable in various medical purposes including surgery, tissue regeneration, burns, traumatic injuries, and 3D cell culture platforms.

Polyelectrolyte-mediated increase of biofilm mass formation.

BACKGROUND: Biofilm formation is associated with various aspects of bacterial and fungal infection. This study was designed to assess the impact of diverse natural polyelectrolytes, such as DNA, F-actin, neurofilaments (NFs), vimentin and purified Pf1 bacteriophage on biofilm formation and swarming motility of select pathogens including Pseudomonas aeruginosa associated with lung infections in CF patients. RESULTS: The bacteriophage Pf1 (1 mg/ml) significantly increased biofilm mass produced by Pseudomonas aeruginosa P14, Escherichia coli RS218 and Bacillus subtilis ATCC6051. DNA, F-actin, NFs and Pf1 also increased biofilm mass of the fungal C. albicans 1409 strain. Addition of F-actin, DNA or Pf1 bacteriophage to 0.5% agar plates increased swarming motility of Pseudomonas aeruginosa Xen5. CONCLUSIONS: The presence of polyelectrolytes at infection sites is likely to promote biofilm growth and bacterial swarming.

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

Effects of non-linearity on cell-ECM interactions.

Filamentous biopolymers such as F-actin, vimentin, fibrin and collagen that form networks within the cytoskeleton or the extracellular matrix have unusual rheological properties not present in most synthetic soft materials that are used as cell substrates or scaffolds for tissue engineering. Gels formed by purified filamentous biopolymers are often strain stiffening, with an elastic modulus that can increase an order of magnitude at moderate strains that are relevant to cell and tissue deformation in vivo. This review summarizes some experimental studies of non-linear rheology in biopolymer gels, discusses possible molecular mechanisms that account for strain stiffening, and explores the possible relevance of non-linear rheology to the interactions between cell and extracellular matrices.

Antibacterial activity of the human host defence peptide LL-37 and selected synthetic cationic lipids against bacteria associated with oral and upper respiratory tract infections.

OBJECTIVES: We aim to develop antibacterial peptide mimics resistant to protease degradation, with broad-spectrum activity at sites of infection. METHODS: The bactericidal activities of LL-37, ceragenins CSA-13, CSA-90 and CSA-92 and the spermine-conjugated dexamethasone derivative D2S were evaluated using MIC and MBC measurements. Gingival fibroblast counting, interleukin-8 (IL-8) and lactate dehydrogenase (LDH) release from keratinocytes (HaCat) were used to determine effects on cell growth, pro-inflammatory response and toxicity. RESULTS: All tested cationic lipids showed stronger bactericidal activity than LL-37. Incubation of Staphylococcus aureus with half the MIC of LL-37 led to the appearance of bacteria resistant to its bactericidal effects, but identical incubations with CSA-13 or D2S did not produce resistant bacteria. Cathelicidin LL-37 significantly increased the total number of gingival fibroblasts, but ceragenins and D2S did not alter gingival fibroblast growth. Cationic lipids showed no toxicity to HaCat cells at concentrations resulting in bacterial killing. CONCLUSIONS: These data suggest that cationic lipids such as ceragenins warrant further testing as potential novel antibacterial agents.

Matching mechanical heterogeneity of the native spinal cord augments axon infiltration in 3D-printed scaffolds.

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often match the anisotropy of native tissue using guidance cues along the rostral-caudal axis, but current approaches do not mimic the heterogeneity of host tissue mechanics. Although white and gray matter have different mechanical properties, it remains unclear whether tissue mechanics also vary along the length of the cord. Mechanical testing performed in this study indicates that bulk spinal cord mechanics do differ along anatomical level and that these differences are caused by variations in the ratio of white and gray matter. These results suggest that scaffolds recreating the heterogeneity of spinal cord tissue mechanics must account for the disparity between gray and white matter. Digital light processing (DLP) provides a means to mimic spinal cord topology, but has previously been limited to printing homogeneous mechanical properties. We describe a means to modify DLP to print scaffolds that mimic spinal cord mechanical heterogeneity caused by variation in the ratio of white and gray matter, which improves axon infiltration compared to controls exhibiting homogeneous mechanical properties. These results demonstrate that scaffolds matching the mechanical heterogeneity of white and gray matter improve the effectiveness of biomaterials transplanted within the injured spinal cord.

Divalent cation-induced cluster formation by polyphosphoinositides in model membranes.

Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.

Divalent cation-dependent formation of electrostatic PIP2 clusters in lipid monolayers.

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP(2)), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP(2), but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP(2) and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP(2) clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP(2) even at small overall concentrations of PIP(2).

Nonlinear elasticity in biological gels.

The mechanical properties of soft biological tissues are essential to their physiological function and cannot easily be duplicated by synthetic materials. Unlike simple polymer gels, many biological materials--including blood vessels, mesentery tissue, lung parenchyma, cornea and blood clots--stiffen as they are strained, thereby preventing large deformations that could threaten tissue integrity. The molecular structures and design principles responsible for this nonlinear elasticity are unknown. Here we report a molecular theory that accounts for strain-stiffening in a range of molecularly distinct gels formed from cytoskeletal and extracellular proteins and that reveals universal stress-strain relations at low to intermediate strains. The input to this theory is the force-extension curve for individual semi-flexible filaments and the assumptions that biological networks composed of these filaments are homogeneous, isotropic, and that they strain uniformly. This theory shows that systems of filamentous proteins arranged in an open crosslinked mesh invariably stiffen at low strains without requiring a specific architecture or multiple elements with different intrinsic stiffness.

Soft materials to treat central nervous system injuries: evaluation of the suitability of non-mammalian fibrin gels.

Polymeric scaffolds formed from synthetic or natural materials have many applications in tissue engineering and medicine, and multiple material properties need to be optimized for specific applications. Recent studies have emphasized the importance of the scaffolds' mechanical properties to support specific cellular responses in addition to considerations of biochemical interactions, material transport, immunogenicity, and other factors that determine biocompatibility. Fibrin gels formed from purified fibrinogen and thrombin, the final two reactants in the blood coagulation cascade, have long been shown to be effective in wound healing and supporting the growth of cells in vitro and in vivo. Fibrin, even without additional growth factors or other components has potential for use in neuronal wound healing in part because of its mechanical compliance that supports the growth of neurons without activation of glial proliferation. This review summarizes issues related to the use of fibrin gels in neuronal cell contexts, with an emphasis on issues of immunogenicity, and considers the potential advantages and disadvantages of fibrin prepared from non-mammalian sources.

Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding.

Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a simple mechanism for driving domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles, which thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium- and copper-crossbridged domains of synthetic block copolymers or natural lipid (phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes and/or spacings that can last for years. Lateral segregation in these 'ligand-responsive Janus assemblies' couples weakly to curvature and proves to be restricted within phase diagrams to narrow regimes of pH and cation concentration that are centred near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for strong lateral segregation shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand-induced pattern formation within self-assemblies.

Nonlinear elasticity of stiff filament networks: strain stiffening, negative normal stress, and filament alignment in fibrin gels.

Many biomaterials formed by cross-linked semiflexible or rigid filaments exhibit nonlinear theology in the form of strain-stiffening and negative normal stress when samples are deformed in simple shear geometry. Two different classes of theoretical models have been developed to explain this nonlinear elastic response, which is neither predicted by rubber elasticity theory nor observed in elastomers or gels formed by flexible polymers. One model considers the response of isotropic networks of semiflexible polymers that have nonlinear force-elongation relations arising from their thermal fluctuations. The other considers networks of rigid filaments with linear force-elongation relations in which nonlinearity arises from nonaffine deformation and a shift from filament bending to stretching at increasing strains. Fibrin gels are a good experimental system to test these theories because the fibrin monomer assembles under different conditions to form either thermally fluctuating protofibrils with persistence length on the order of the network mesh size, or thicker rigid fibers. Comparison of rheologic and optical measurements shows that strain stiffening and negative normal stress appear at smaller strains than those at which filament orientation is evident from birefringence. Comparisons of shear to normal stresses and the strain-dependence of shear moduli and birefringence suggest methods to evaluate the applicability of different theories of rod-like polymer networks. The strain-dependence of the ratio of normal stress to shear stress is one parameter that distinguishes semiflexible and rigid filament models, and comparisons with experiments reveal conditions under which specific theories may be applicable.

Fibrin gels and their clinical and bioengineering applications.

Fibrin gels, prepared from fibrinogen and thrombin, the key proteins involved in blood clotting, were among the first biomaterials used to prevent bleeding and promote wound healing. The unique polymerization mechanism of fibrin, which allows control of gelation times and network architecture by variation in reaction conditions, allows formation of a wide array of soft substrates under physiological conditions. Fibrin gels have been extensively studied rheologically in part because their nonlinear elasticity, characterized by soft compliance at small strains and impressive stiffening to resist larger deformations, appears essential for their function as haemostatic plugs and as matrices for cell migration and wound healing. The filaments forming a fibrin network are among the softest in nature, allowing them to deform to large extents and stiffen but not break. The biochemical and mechanical properties of fibrin have recently been exploited in numerous studies that suggest its potential for applications in medicine and bioengineering.

Salivary mucins inhibit antibacterial activity of the cathelicidin-derived LL-37 peptide but not the cationic steroid CSA-13.

OBJECTIVES: Cationic antimicrobial peptides (CAPs) are the effector molecules of innate immunity, similar in potency to classic antibiotics that function in the first-line of defence against infectious agents. The purpose of this study was to investigate the effects of negatively charged mucins on the antibacterial activity of the positively charged cathelicidin LL-37 peptide, its synthetic analogue WLBU2 and the antimicrobial cationic steroid CSA-13. METHODS: Mucin, DNA, F-actin and hCAP-18/LL-37 in saliva samples were evaluated by microscopy or immunoblotting. Bacterial killing assays and determination of MICs were used to determine bactericidal activity. Binding of rhodamine-B-labelled LL-37 peptide to mucin was fluorimetrically assessed. RESULTS: Microscopic evaluation of saliva after addition of rhodamine-B-labelled LL-37 showed localization similar to that observed after the addition of a specific mucin-binding lectin. Immunoblotting confirmed the presence of hCAP-18/LL-37 in saliva samples and LL-37 peptide bound to isolated submaxillary gland mucin-coated plates. Mucin/LL-37 binding was partially prevented by treatment of mucin with neuraminidase, indicating involvement of sialic acid moieties. Decreased LL-37 and WLBU2 antibacterial activity was observed in the presence of mucin or dialysed human saliva, whereas CSA-13 antibacterial activity was significantly resistant to inhibition by mucins. CONCLUSIONS: This study shows that the antibacterial LL-37 peptide and its synthetic analogue WLBU2 are inhibited by salivary mucin and that the cationic steroid CSA-13 retains most of its function in the presence of an equal amount of mucin or saliva.

Control of cell morphology and differentiation by substrates with independently tunable elasticity and viscous dissipation.

The mechanical properties of extracellular matrices can control the function of cells. Studies of cellular responses to biomimetic soft materials have been largely restricted to hydrogels and elastomers that have stiffness values independent of time and extent of deformation, so the substrate stiffness can be unambiguously related to its effect on cells. Real tissues, however, often have loss moduli that are 10 to 20% of their elastic moduli and behave as viscoelastic solids. The response of cells to a time-dependent viscous loss is largely uncharacterized because appropriate viscoelastic materials are lacking for quantitative studies. Here we report the synthesis of soft viscoelastic solids in which the elastic and viscous moduli can be independently tuned to produce gels with viscoelastic properties that closely resemble those of soft tissues. Systematic alteration of the hydrogel viscosity demonstrates the time dependence of cellular mechanosensing and the influence of viscous dissipation on cell phenotype.

Uncoupling shear and uniaxial elastic moduli of semiflexible biopolymer networks: compression-softening and stretch-stiffening.

Gels formed by semiflexible filaments such as most biopolymers exhibit non-linear behavior in their response to shear deformation, e.g., with a pronounced strain stiffening and negative normal stress. These negative normal stresses suggest that networks would collapse axially when subject to shear stress. This coupling of axial and shear deformations can have particularly important consequences for extracellular matrices and collagenous tissues. Although measurements of uniaxial moduli have been made on biopolymer gels, these have not directly been related to the shear response. Here, we report measurements and simulations of axial and shear stresses exerted by a range of hydrogels subjected to simultaneous uniaxial and shear strains. These studies show that, in contrast to volume-conserving linearly elastic hydrogels, the Young's moduli of networks formed by the biopolymers are not proportional to their shear moduli and both shear and uniaxial moduli are strongly affected by even modest degrees of uniaxial strain.