Sodium hyaluronate-g-2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide with enhanced affinity towards MMP12 catalytic domain to be used as visco-supplement with increased degradation resistance.

The present paper describes the functionalization of sodium hyaluronate (NaHA) with a small molecule (2-((N-(6-aminohexyl)-4-methoxyphenyl)sulfonamido)-N-hydroxyacetamide) (MMPI) having proven inhibitory activity against membrane metalloproteins involved in inflammatory processes (i.e. MMP12). The obtained derivative (HA-MMPI) demonstrated an increased resistance to the in-vitro degradation by hyaluronidase, viscoelastic properties close to those of healthy human synovial fluid, cytocompatibility towards human chondrocytes and nanomolar affinity towards MMP 12. Thus, HA-MMPI can be considered a good candidate as viscosupplement in the treatment of knee osteoarticular disease.

Types of biomaterials useful in brain repair.

Biomaterials is an emerging field in the study of brain tissue engineering and repair or neurogenesis. The fabrication of biomaterials that can replicate the mechanical and viscoelastic features required by the brain, including the poroviscoelastic responses, force dissipation, and solute diffusivity are essential to be mapped from the macro to the nanoscale level under physiological conditions in order for us to gain an effective treatment for neurodegenerative diseases. This research topic has identified a critical study gap that must be addressed, and that is to source suitable biomaterials and/or create reliable brain-tissue-like biomaterials. This chapter will define and discuss the various types of biomaterials, their structures, and their function-properties features which would enable the development of next-generation biomaterials useful in brain repair.

Viscoelastic control of spatiotemporal order in bacterial active matter.

Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.

Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes.

Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley's K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Modelling the microbial dynamics and antimicrobial resistance development of Listeria in viscoelastic food model systems of various structural complexities.

Minimal processing for microbial decontamination, such as the use of natural antimicrobials, is gaining interest in the food industry as these methods are generally milder than conventional processing, therefore better maintaining the nutritional content and sensory characteristics of food products. The aim of this study was to quantify the impact of (i) structural composition and complexity, (ii) growth location and morphology, and (iii) the natural antimicrobial nisin, on the microbial dynamics of Listeria innocua. More specifically, viscoelastic food model systems of various compositions and internal structure were developed and characterised, i.e. monophasic Xanthan gum-based and biphasic Xanthan gum/Whey protein-based viscoelastic systems. The microbial dynamics of L. innocua at 10 °C, 30 °C and 37 °C were monitored and compared for planktonic growth in liquid, or in/on (immersed or surface colony growth) the developed viscoelastic systems, with or without a sublethal concentration of nisin. Microscopy imaging was used to determine the bacterial colony size and spatial organisation in/on the viscoelastic systems. Selective growth of L. innocua on the protein phase of the developed biphasic system was observed for the first time. Additionally, significant differences were observed in the colony size and distribution in the monophasic Xanthan gum-based systems depending on (i) the type of growth (surface/immersed) and (ii) the Xanthan gum concentration. Furthermore, the system viscosity in monophasic Xanthan gum-based systems had a protective role against the effects of nisin for immersed growth, and a further inhibitory effect for surface growth at a suboptimal temperature (10 °C). These findings give a systematic quantitative insight on the impact of nisin as an environmental challenge on the growth and spatial organisation of L. innocua, in viscoelastic food model systems of various structural compositions/complexities. This study highlights the importance of accounting for system structural composition/complexity when designing minimal food processing methods with natural antimicrobials.

Viscoelastic modeling of the fusion of multicellular tumor spheroids in growth phase.

BACKGROUND: Since several decades, the experiments have highlighted the analogy of fusing cell aggregates with liquid droplets. The physical macroscopic models have been derived under incompressible assumptions. The aim of this paper is to provide a 3D model of growing spheroids, which is more relevant regarding embryo cell aggregates or tumor cell spheroids. METHODS: We extend the past approach to a compressible 3D framework in order to account for the tumor spheroid growth. We exhibit the crucial importance of the effective surface tension, and of the inner pressure of the spheroid to describe precisely the fusion. The experimental data were obtained on spheroids of colon carcinoma human cells (HCT116 cell line). After 3 or 6 days of culture, two identical spheroids were transferred in one well and their fusion was monitored by live videomicroscopy acquisition each 2 h during 72 h. From these images the neck radius and the diameter of the assembly of the fusing spheroids are extracted. RESULTS: The numerical model is fitted with the experiments. It is worth noting that the time evolution of both neck radius and spheroid diameter are quantitatively obtained. The interesting feature lies in the fact that such measurements characterise the macroscopic rheological properties of the tumor spheroids. CONCLUSIONS: The experimental determination of the kinetics of neck radius and overall diameter during spheroids fusion characterises the rheological properties of the spheroids. The consistency of the model is shown by fitting the model with two different experiments, enhancing the importance of both surface tension and cell proliferation. GENERAL SIGNIFICANCE: The paper sheds new light on the macroscopic rheological properties of tumor spheroids. It emphasizes the role of the surface tension and the inner pressure in the fusion of growing spheroid. Under geometrical assumptions, the model reduces to a 2-parameter differential equation fit with experimental measurements. The 3-D partial differential system makes it possible to study the fusion of spheroids in non-symmetrical or more general frameworks.

A Versatile Framework for Simulating the Dynamic Mechanical Structure of Cytoskeletal Networks.

Computer simulations can aid in understanding how collective materials properties emerge from interactions between simple constituents. Here, we introduce a coarse-grained model that enables simulation of networks of actin filaments, myosin motors, and cross-linking proteins at biologically relevant time and length scales. We demonstrate that the model qualitatively and quantitatively captures a suite of trends observed experimentally, including the statistics of filament fluctuations, and mechanical responses to shear, motor motilities, and network rearrangements. We use the simulation to predict the viscoelastic scaling behavior of cross-linked actin networks, characterize the trajectories of actin in a myosin motility assay, and develop order parameters to measure contractility of a simulated actin network. The model can thus serve as a platform for interpretation and design of cytoskeletal materials experiments, as well as for further development of simulations incorporating active elements.

Uniform Contraction-Expansion Description of Relative Centromere and Telomere Motion.

Internal organization and dynamics of the eukaryotic nucleus have been at the front of biophysical research in recent years. It is believed that both dynamics and location of chromatin segments are crucial for genetic regulation. Here we study the relative motion between centromeres and telomeres at various distances and at times relevant for genetic activity. Using live-imaging fluorescent microscopy coupled to stochastic analysis of relative trajectories, we find that the interlocus motion is distance-dependent with a varying fractional memory. In addition to short-range constraining, we also observe long-range anisotropic-enhanced parallel diffusion, which contradicts the expectation for classic viscoelastic systems. This motion is linked to uniform expansion and contraction of chromatin in the nucleus, and leads us to define and measure a new (to our knowledge) uniform contraction-expansion diffusion coefficient that enriches the contemporary picture of nuclear behavior. Finally, differences between loci types suggest that different sites along the genome experience distinctive coupling to the nucleoplasm environment at all scales.

Evaluation of a new method of irrigation and aspiration for removal of ophthalmic viscoelastic device during cataract surgery in a porcine model.

BACKGROUND: To determine if a method for irrigation and aspiration (I/A) during cataract surgery provides effective removal of ophthalmic viscoelastic device (OVD). METHODS: Japanese porcine eyes were used to evaluate I/A performance with Technique 1 (the I/A tip placed on the center of the anterior surface of the IOL), Technique 2 (the I/A tip alternately pressed near the edge of the IOL optic anterior surface on one side and then the other to tilt the IOL back and forth), and Technique 3 (the I/A tip inserted behind the IOL optic, between it and the posterior capsule). Techniques 1 and 2 were compared using the Miyake-Apple posterior view video technique to visualize the flow of irrigation fluid containing triamcinolone acetonide particles behind the IOL. To check the efficacy of OVD removal from behind the IOL for of all three I/A techniques, OVD with fluorescein beads were inserted inside the lens capsule before implantation of the IOL. After each I/A technique, eyes were prepared for Miyake-Apple viewing and pictures of the lens capsule were taken using fluorescent microscopy. Residual fluorescein beads in the capsular bag were analyzed. RESULTS: Technique 1 resulted in a straight flow of fluid behind the IOL, while Technique 2 resulted in a vortex flow. The average amount of OVD retained inside the capsule after using Technique 2 or 3 was significantly lower than after using Technique 1 (p <0.0001). CONCLUSIONS: Technique 2 proved to remove more effectively fluorescein bead-labelled OVD under the IOL than Technique 1.

A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.

A novel, simple, and reproducible method for cultivating pathological tissues obtained from human eyes during surgery was developed using viscoelastic material as a tissue adherent to facilitate cell attachment and expansion and calcium imaging of cultured cells challenged by mechanical and acetylcholine (ACh) stimulation as well as inflammatory studies. Anterior lens capsule-lens epithelial cells (aLC-LECs) from cataract surgery and proliferative diabetic retinopathy (PDR) fibrovascular epiretinal membranes (fvERMs) from human eyes were used in the study. We hereby show calcium signaling in aLC-LECs by mechanical and acetylcholine (ACh) stimulation and indicate presence of ACh receptors in these cells. Furthermore, an ex vivo study model was established for measuring the inflammatory response in fvERMs and aLC-LECs upon TNFα treatment.

Influence of molecular motors on the motion of particles in viscoelastic media.

We study theoretically and by numerical simulations the motion of particles driven by molecular motors in a viscoelastic medium representing the cell cytoplasm. For this, we consider a generalized Langevin equation coupled to a stochastic stepping dynamics for the motors that takes into account the action of each motor separately. In the absence of motors, the model produces subdiffusive motion of particles characterized by a power-law scaling of the mean square displacement versus the lag time as t^{α}, with 0<α<1, similar to that observed in cells. Our results show how the action of the motors can induce a transition to a superdiffusive regime at large lag times with the characteristics of those found in experiments reported in the literature. We also show that at small lag times, the motors can act as static crosslinkers that slow down the natural subdiffusive transport. An analysis of previously reported experimental data in the relevant time scales provides evidence of this phenomenon. Finally, we study the effect of a harmonic potential representing an optical trap, and we show a way to approach to a macroscopic description of the active transport in cells. This last point stresses the relevance of the molecular motors for generating not only directed motion to specific targets, but also fast diffusivelike random motion.

Interactions of glycosphingolipids and lipopolysaccharides with silica and polyamide surfaces: adsorption and viscoelastic properties.

Bacterial outer membrane components play a critical role in bacteria-surface interactions (adhesion and repulsion). Sphingomonas species (spp.) differ from other Gram-negative bacteria in that they lack lipopolysaccharides (LPSs) in their outer membrane. Instead, Sphingomonas spp. outer membrane consists of glycosphingolipids (GSLs). To delineate the properties of the outer membrane of Sphingomonas spp. and to explain the adhesion of these cells to surfaces, we employed a single-component-based approach of comparing GSL vesicles to LPS vesicles. This is the first study to report the formation of vesicles containing 100% GSL. Significant physicochemical differences between GSL and LPS vesicles are reported. Composition-dependent vesicle adherence to different surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D) technology was observed, where higher GSL content resulted in higher mass accumulation on the sensor. Additionally, the presence of 10% GSL and above was found to promote the relative rigidity of the vesicle obtaining viscoelastic ratio of 30-70% higher than that of pure LPS vesicles.

A new highly viscoelastic hyaluronic acid gel: rheological properties, biocompatibility and clinical investigation in esthetic and restorative surgery.

Nowadays there is an increased demand for safe and effective volume enhancing fillers to achieve soft tissue augmentation in order to overcome tissue defects and aging-associated skin changes. In the present study we characterized the rheological and biological properties of Variofill(®), a new highly viscoelastic hyaluronic acid gel, by investigating the local effects following subcutaneous implantation in the rat to detect the host-tissue reactions and biodegradation over 18 months. We also investigated, for the first time, the application of Variofill(®) in esthetic and restorative surgery in two medical case reports. In the first case report we successfully performed Variofill(®) treatment to improve facial scars in a patient previously involved in a car crash. In the second case report we carried out a novel procedure involving a high-dose (1000 ml) injection of Variofill(®) into the dermis and subcutis of the abdominal quadrants in order to allow a classic reconstructive procedure of the abdominal wall in a patient presenting a wide incisional hernia.

Interfacial dilatational viscoelasticity of human meibomian lipid films.

PURPOSE/AIM: The meibomian lipid layer is able to withstand the enormous stresses and deformations that occur during blinking due to the combination of its elastic and viscous properties. The purpose of this study was to measure the dilatational viscoelasticity of in vitro meibomian lipid films and compare how these properties differ between room temperature and physiological temperatures. Viscoelasticity was also compared with meibomian lipid films seeded with cholesterol or ß-carotene (the levels of these lipid species change in disease states). MATERIALS AND METHODS: Dilatational viscoelasticity (E) was measured using an oscillating pendant drop method. Measurements were carried out on spread films at the air-water interface as a function of frequency (0.1256-12.56 rad/s) at various temperatures between 18-43 °C. RESULTS: Generally, E gradually decreased as the overall temperature was increased. At both 37 and 20 °C, films demonstrated that the elastic modulus (E') was more dominant than the viscous modulus (E″), indicating films were more solid-like than fluid-like, regardless of temperature. E' and E″ were also dependant on frequency, indicating some molecular rearrangements of the lipid molecules as films were compressed and expanded. Films seeded with cholesterol or ß-carotene showed a modest increase in the moduli. CONCLUSIONS: These results are consistent with previous findings which have predicted and indicated that the meibomian lipid layer is a viscoelastic film at the air-liquid interface. These properties are integral to how the tear film lipid layer is able to maintain its structure, and hence integrity of the ocular surface.

Effect of cut rates on fluidic behavior of chopped vitreous.

PURPOSE: To analyze the viscoelastic properties of the chopped vitreous at different cut rates to better understand complex fluidic behavior of chopped vitreous during vitrectomy. METHODS: Twenty- and 25-gauge cutters were used to cut 107 porcine eyes at different cut rates of 500, 1000, 1500, 2000, and 2500 cuts per minute with a fixed vacuum pressure of 500 mmHg. Each sample was immediately tested using a shear rheometer to obtain its rheologic properties. RESULTS: Chopped vitreous demonstrated significantly lower viscosity (0.039 ± 0.01 Pa·s) than intact vitreous (908.1 ± 210.8 Pa·s). However, cut rate did not have any significant impact on viscosity. In addition, chopped vitreous presented elastic behavior. It was shown that the compliance, the inverse of stiffness, of chopped vitreous is much higher than that of intact vitreous (1.83 ± 0.31 Pa for intact vitreous and 85.3 ± 14.4 Pa for chopped vitreous) and varies in a nonlinear fashion when cut at different cut rates. CONCLUSION: Cut rate affects the rheologic properties of the chopped vitreous and, therefore, its flow inside the vitrectomy system. It is essential to account for both viscosity and elasticity of chopped vitreous to understand flow behavior during vitrectomy.

Effects of temperature and trehalose on foam separation of nisin from the culture broth produced by Lactococcus lactis subspecies lactis W28.

Nisin is an antimicrobial peptide, an important biopreservative, and it is produced by certain strains of Lactococcus lactis ssp. lactis. In this paper, a foam separation technique was used for the separation of nisin from its culture broth, and the effects of temperature and trehalose on the performance of foam separation of nisin were studied to increase the enrichment ratio and recovery percentage of nisin and decrease the inactivity percentage of nisin. The results showed that temperature and trehalose significantly affected the performance of foam separation of nisin. Under the optimum conditions of 50°C temperature, 150-mL/min air flow rate, 400-mL initial loading liquid volume, and 1-g/L trehalose concentration, the maximum enrichment ratio, recovery percentage, and the minimum inactivity percentage of nisin reached 23.7, 84.1%, and 5.9%, respectively, which were, respectively, 5.04, 0.93, and 1.03 times more than those under the conditions of 20°C temperature, 150-mL/min air flow rate, 400-mL initial loading liquid volume, and no trehalose addition. These results indicated that the change of temperature and the addition of trehalose could improve the performance of foam separation of nisin.

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

Comparative study of the viscoelastic mechanical behavior of agarose and poly(ethylene glycol) hydrogels.

This study presents a comparative investigation into differences in the mechanical properties between two hydrogels commonly used in cartilage tissue engineering [agarose vs. poly(ethylene glycol) (PEG)], but which are formed through distinctly different crosslinking mechanisms (physical vs. covalent, respectively). The effects of hydrogel chemistry, precursor concentration, platen type (nonporous vs. porous) used in compression bioreactors, and degradation (for PEG) on the swelling properties and static and dynamic mechanical properties were examined. An increase in precursor concentration resulted in decreased equilibrium mass swelling ratios but increased equilibrium moduli and storage moduli for both hydrogels (p < 0.05). Agarose displayed large stress relaxations and a frequency dependence indicating its viscoelastic properties. Contrarily, PEG hydrogels displayed largely elastic behavior with minimal stress relaxation and frequency dependence. In biodegradable PEG hydrogels, the largely elastic behavior was retained during degradation. The type of platen did not affect static mechanical properties, but porous platens led to a reduced storage modulus for both hydrogels implicating fluid flow. In summary, agarose and PEG exhibit vastly different mechanical behaviors; a finding largely attributed to differences in their chemistries and fluid movement. Taken together, these design choices (hydrogel chemistry/structure, loading conditions) will likely have a profound effect on the tissue engineering outcome.