Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

Isolation of Mixed Compositions of Cellulose Nanocrystals, Microcrystalline Cellulose, and Lignin Nanoparticles from Wood Pulps.

From a circular economy perspective, one-pot strategies for the isolation of cellulose nanomaterials at a high yield and with multifunctional properties are attractive. Here, the effects of lignin content (bleached vs unbleached softwood kraft pulp) and sulfuric acid concentration on the properties of crystalline lignocellulose isolates and their films are explored. Hydrolysis at 58 wt % sulfuric acid resulted in both cellulose nanocrystals (CNCs) and microcrystalline cellulose at a relatively high yield (>55%), whereas hydrolysis at 64 wt % gave CNCs at a lower yield (<20%). CNCs from 58 wt % hydrolysis were more polydisperse and had a higher average aspect ratio (1.5-2×), a lower surface charge (2×), and a higher shear viscosity (100-1000×). Hydrolysis of unbleached pulp additionally yielded spherical nanoparticles (NPs) that were <50 nm in diameter and identified as lignin by nanoscale Fourier transform infrared spectroscopy and IR imaging. Chiral nematic self-organization was observed in films from CNCs isolated at 64 wt % but not from the more heterogeneous CNC qualities produced at 58 wt %. All films degraded to some extent under simulated sunlight trials, but these effects were less pronounced in lignin-NP-containing films, suggesting a protective feature, but the hemicellulose content and CNC crystallinity may be implicated as well. Finally, heterogeneous CNC compositions obtained at a high yield and with improved resource efficiency are suggested for specific nanocellulose uses, for instance, as thickeners or reinforcing fillers, representing a step toward the development of application-tailored CNC grades.

Surfactant-Free Stabilization of Aqueous Graphene Dispersions Using Starch as a Dispersing Agent.

Attention to graphene dispersions in water with the aid of natural polymers is increasing with improved awareness of sustainability. However, the function of biopolymers that can act as dispersing agents in graphene dispersions is not well understood. In particular, the use of starch to disperse pristine graphene materials deserves further investigation. Here, we report the processing conditions of aqueous graphene dispersions using unmodified starch. We have found that the graphene content of the starch-graphene dispersion is dependent on the starch fraction. The starch-graphene sheets are few-layer graphene with a lateral size of 3.2 µm. Furthermore, topographical images of these starch-graphene sheets confirm the adsorption of starch nanoparticles with a height around 5 nm on the graphene surface. The adsorbed starch nanoparticles are ascribed to extend the storage time of the starch-graphene dispersion up to 1 month compared to spontaneous aggregation in a nonstabilized graphene dispersion without starch. Moreover, the ability to retain water by starch is reduced in the presence of graphene, likely due to environmental changes in the hydroxyl groups responsible for starch-water interactions. These findings demonstrate that starch can disperse graphene with a low oxygen content in water. The aqueous starch-graphene dispersion provides tremendous opportunities for environmental-friendly packaging applications.

Milling induced amorphisation and recrystallization of α-lactose monohydrate.

Preprocessing of pharmaceutical powders is a common procedure to condition the materials for a better manufacturing performance. However, such operations may induce undesired material properties modifications when conditioning particle size through milling, for example. Modification of both surface and bulk material structure will change the material properties, thus affecting the processability of the powder. Hence it is essential to control the material transformations that occur during milling. Topographical and mechanical changes in surface properties can be a preliminary indication of further material transformations. Therefore a surface evaluation of the α-lactose monohydrate after short and prolonged milling times has been performed. Unprocessed α-lactose monohydrate and spray dried lactose were evaluated in parallel to the milled samples as reference examples of the crystalline and amorphous lactose structure. Morphological differences between unprocessed α-lactose, 1 h and 20 h milled lactose and spray dried lactose were detected from SEM and AFM images. Additionally, AFM was used to simultaneously characterize particle surface amorphicity by measuring energy dissipation. Extensive surface amorphicity was detected after 1 h of milling while prolonged milling times showed only a moderate particle surface amorphisation. Bulk material characterization performed with DSC indicated a partial amorphicity for the 1 h milled lactose and a fully amorphous thermal profile for the 20 h milled lactose. The temperature profiles however, were shifted somewhat in the comparison to the amorphous reference, particularly after extended milling, suggesting a different amorphous state compared to the spray-dried material. Water loss during milling was measured with TGA, showing lower water content for the lactose amorphized through milling compared to spray dried amorphous lactose. The combined results suggest a surface-bulk propagation of the amorphicity during milling in combination with a different amorphous structural conformation to that of the amorphous spray dried lactose. The hardened surface may be due to either surface crystallization of lactose or to formation of a low-water glass transition.

AFM Colloidal Probe Measurements Implicate Capillary Condensation in Punch-Particle Surface Interactions during Tableting.

Adhesion of the powders to the punches is a common issue during tableting. This phenomenon is known as sticking and affects the quality of the manufactured tablets. Defective tablets increase the cost of the manufacturing process. Thus, the ability to predict the tableting performance of the formulation blend before the process is scaled-up is important. The adhesive propensity of the powder to the tableting tools is mostly governed by the surface-surface adhesive interactions. Atomic force microscopy (AFM) colloidal probe is a surface characterization technique that allows the measurement of the adhesive interactions between two materials of interest. In this study, AFM steel colloidal probe measurements were performed on ibuprofen, MCC (microcrystalline cellulose), α-lactose monohydrate, and spray-dried lactose particles as an approach to modeling the punch-particle surface interactions during tableting. The excipients (lactose and MCC) showed constant, small, attractive, and adhesive forces toward the steel surface after a repeated number of contacts. In comparison, ibuprofen displayed a much larger attractive and adhesive interaction increasing over time both in magnitude and in jump-in/jump-out separation distance. The type of interaction acting on the excipient-steel interface can be related to a van der Waals force, which is relatively weak and short-ranged. By contrast, the ibuprofen-steel interaction is described by a capillary force profile. Even though ibuprofen is not highly hydrophilic, the relatively smooth surfaces of the crystals allow "contact flooding" upon contact with the steel probe. Capillary forces increase because of the "harvesting" of moisture-due to the fast condensation kinetics-leaving a residual condensate that contributes to increase the interaction force after each consecutive contact. Local asperity contacts on the more hydrophilic surface of the excipients prevent the flooding of the contact zone, and there is no such adhesive effect under the same ambient conditions. The markedly different behavior detected by force measurements clearly shows the sticky and nonsticky propensity of the materials and allows a mechanistic description.

Determination of Interfacial Amorphicity in Functional Powders.

The nature of the surfaces of particles of pharmaceutical ingredients, food powders, and polymers is a determining factor for their performance in for example tableting, powder handling, or mixing. Changes on the surface structure of the material will impact the flow properties, dissolution rate, and tabletability of the powder blend. For crystalline materials, surface amorphization is a phenomenon which is known to impact performance. Since it is important to measure and control the level of amorphicity, several characterization techniques are available to determine the bulk amorphous content of a processed material. The possibility of characterizing the degree of amorphicity at the surface, for example by studying the mechanical properties of the particles' surface at the nanoscale, is currently only offered by atomic force microscopy (AFM). The AFM PeakForce QNM technique has been used to measure the variation in energy dissipation (eV) at the surface of the particles which sheds light on the mechanical changes occurring as a result of amorphization or recrystallization events. Two novel approaches for the characterization of amorphicity are presented here. First, since particles are heterogeneous, we present a methodology to present the results of extensive QNM analysis of multiple particles in a coherent and easily interpreted manner, by studying cumulative distributions of dissipation data with respect to a threshold value which can be used to distinguish the crystalline and amorphous states. To exemplify the approach, which is generally applicable to any material, reference materials of purely crystalline α-lactose monohydrate and completely amorphous spray dried lactose particles were compared to a partially amorphized α-lactose monohydrate sample. Dissipation data are compared to evaluations of the lactose samples with conventional AFM and SEM showing significant topographical differences. Finally, the recrystallization of the surface amorphous regions in response to humidity was followed by studying the dissipation response of a well-defined surface region over time, which confirms both that dissipation measurement is a useful measure of surface amorphicity and that significant recrystallization occurs at the surface in response to humidity.

Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts.

In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively.

Nanomechanical properties of human skin and introduction of a novel hair indenter.

The mechanical resistance of the stratum corneum, the outermost layer of skin, to deformation has been evaluated at different length scales using Atomic Force Microscopy. Nanomechanical surface mapping was first conducted using a sharp silicon tip and revealed that Young׳s modulus of the stratum corneum varied over the surface with a mean value of about 0.4GPa. Force indentation measurements showed permanent deformation of the skin surface only at high applied loads (above 4µN). The latter effect was further demonstrated using nanomechanical imaging in which the obtained depth profiles clearly illustrate the effects of increased normal force on the elastic/plastic surface deformation. Force measurements utilizing the single hair fiber probe supported the nanoindentation results of the stratum corneum being highly elastic at the nanoscale, but revealed that the lateral scale of the deformation determines the effective elastic modulus.This result resolves the fact that the reported values in the literature vary greatly and will help to understand the biophysics of the interaction of razor cut hairs that curl back during growth and interact with the skin.

Tablet mechanics depend on nano and micro scale adhesion, lubrication and structure.

Tablets are the most convenient form for drug administration. However, despite the ease of manufacturing problems such as powder adhesion occur during the production process. This study presents surface and structural characterization of tablets formulated with commonly used excipients (microcrystalline cellulose (MCC), lactose, mannitol, magnesium (Mg) stearate) pressed under different compaction conditions. Tablet surface analyses were performed with scanning electron microscopy (SEM), profilometry and atomic force microscopy (AFM). The mechanical properties of the tablets were evaluated with a tablet hardness test. Local adhesion detected by AFM decreased when Mg stearate was present in the formulation. Moreover, the tablet strength of plastically deformable excipients such as MCC was significantly decreased after addition of Mg stearate. Combined these facts indicate that Mg stearate affects the particle-particle bonding and thus elastic recovery. The MCC excipient also displayed the highest hardness which is characteristic for a highly cohesive material. This is discussed in the view of the relatively high adhesion found between MCC and a hydrophilic probe at the nanoscale using AFM. In contrast, the tablet strength of brittle materials like lactose and mannitol is unaffected by Mg stearate. Thus fracture occurs within the excipient particles and not at particle boundaries, creating new surfaces not previously exposed to Mg stearate. Such uncoated surfaces may well promote adhesive interactions with tools during manufacture.

Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness.

Oncogenes deregulate fundamental cellular functions, which can lead to development of tumors, tumor-cell invasion, and metastasis. As the mechanical properties of cells govern cell motility, we hypothesized that oncogenes promote cell invasion by inducing cytoskeletal changes that increase cellular stiffness. We show that the oncogenes simian virus 40 large T antigen, c-Myc, and cyclin E induce spatial reorganization of the vimentin intermediate filament network in cells. At the cellular level, this reorganization manifests as increased width of vimentin fibers and the collapse of the vimentin network. At nanoscale resolution, the organization of vimentin fibers in these oncogene-expressing cells was more entangled, with increased width of the fibers compared with control cells. Expression of these oncogenes also resulted in up-regulation of the tubulin deacetylase histone deacetylase 6 (HDAC6) and altered spatial distribution of acetylated microtubules. This oncogene expression also induced increases in cellular stiffness and promoted the invasive capacity of the cells. Importantly, HDAC6 was required and sufficient for the structural collapse of the vimentin filament network, and was required for increased cellular stiffness of the oncogene-expressing cells. Taken together, these data are consistent with the possibility that oncogenes can induce cellular stiffness via an HDAC6-induced reorganization of the vimentin intermediate filament network.

Nanobiocomposite adhesion: role of graft length and temperature in a hybrid biomimetic approach.

Cellulose microspheres bearing poly(ε-caprolactone) grafts of different molecular weights were investigated to evaluate the effect of graft length on the interfacial properties. Surface force and friction measurements were performed using an atomic force microscope in colloidal probe mode. The maximum interaction distance and adhesion is dependent on the temperature and the time in contact via a diffusion controlled mechanism. The effects are highest for the longer grafts, and molecular weight thresholds were found to lie between 21 and 34 kDa at 25 °C and between 9 and 21 kDa at 40 °C. The interpenetration of the graft into a matrix leads to "hidden length" contributions to adhesion, analogous to those in natural biocomposites. The nanotribology results display Amontonian behavior, and the friction force at zero applied load is higher at the graft-graft interface than for a bare cellulose sphere interacting with the graft. These results clearly demonstrate the benefits of the grafted polymer layer on the adhesion, toughness, and resistance to shear in the design of cellulosic nanobiocomposites.

Tunable nanolubrication between dual-responsive polyionic grafts.

This study reports on a direct approach of quantitatively probing the nanotribological response of chemically end-grafted polyions. A combination of a quartz crystal microbalance with dissipation and atomic force microscopy, in the now well established colloidal probe mode, was utilized to investigate the stimuli-induced lubrication behavior between poly(2-(dimethylamino)ethyl methacrylate) grafts on gold. Force and friction measurements showed reversible transitions of up to an order of magnitude difference induced by varying the solvent conditions. The greatly enhanced lubrication observed at low pH was attributed to the formation of a repulsive, highly charged, hydrated cushion. At high pH the friction was significantly increased. The system turned attractive above the lower critical solution temperature with a small friction reduction interpreted as being due to nanoscopic flattening at the interfacial boundary.

Force pulling of single cellulose chains at the crystalline cellulose-liquid interface: a molecular dynamics study.

Pulling single cellulose molecules from a crystalline cellulose surface has been modeled by molecular dynamics (MD) simulations of the experimental procedure used in atomic force microscopy (AFM). Specifically, the aim of the study was to investigate cellulose interactions at desorption. Simulations were performed in both water and the organic solvent cyclohexane. Moreover, the effects of initial octamer conformation and orientation with respect to the surface chains were studied. A strong effect from the solvent was observed. In cyclohexane, normal forces of 200-500 pN and energies of 43.5+/-6.0 kJ/mol glucose unit were required to pull off the octamer. The normal forces in water were substantially lower, around 58 pN, and the energies were 18.2+/-3.6 kJ/mol glucose unit. In addition, the lateral components of the pull-off force were shown to provide information on initial conformation and orientation. Hydrogen bonds between the octamer and surface were analyzed and found to be an important factor in the pull-off behavior. Altogether, it was shown that MD provides detailed information on the desorption processes that may be useful for the interpretation of AFM experiments.

Mediation of the nanotribological properties of cellulose by chitosan adsorption.

Cellulosic model surfaces functionalized with chitosan, a naturally occurring cationic biomacromolecule, by in situ adsorption have been studied with an atomic force microscope (AFM) in colloidal probe configuration. The interaction forces on approach and separation, as well as the nanotribological properties, were shown to be highly pH-dependent, and a significant difference in the behavior was seen before and after chitosan adsorption. In general, all forces on approach showed a highly repulsive interaction at shorter distances due to deformation of the probe. At high pH, before chitosan adsorption, a long-range electrostatic repulsion was observed, consistent with DLVO theory. However, at low pH no electrostatic contribution was found before adsorption, probably due to charge neutralization of carboxyl groups. After chitosan adsorption, repulsive forces acting over a much longer distance than predicted by DLVO theory were present at low pH. This effect was ascribed to chain extension of the chitosan species of which the magnitude and the range of the force increased dramatically with higher charge at low pH. In all cases, a typical saw-tooth patterned adhesion was present, with pull-off events occurring at different separations. The frequency of these events after chitosan adsorption was greatly increased at longer distances. Additionally, the adsorbed chitosan markedly reduced the friction, where the largest effect was a 7-fold decrease of the friction coefficient observed at low pH.

Adhesion dynamics for cellulose nanocomposites.

The efficiency of poly(ε-caprolactone) (PCL) as a matrix polymer for cellulose nanocomposites has been investigated at the macromolecular contact level using atomic force microscopy in a colloidal probe configuration. Model cellulose microspheres grafted with PCL were prepared via ring-opening polymerization. Force measurements between the functionalized particles revealed the adhesion to be highly dependent on the contact time because of a diffusion-controlled mechanism. Moreover, an increase of the temperature to 60 degrees C (close to T(m) for the PCL graft) greatly enhanced the adhesion at the polymer-polymer interface, demonstrating the importance of entanglements in the annealing of composite materials.

Top-down grafting of xyloglucan to gold monitored by QCM-D and AFM: enzymatic activity and interactions with cellulose.

This study focuses on the manufacture and characterization of model surfaces consisting of end-grafted xyloglucan (XG), a naturally occurring polysaccharide, onto a gold substrate. The now well-established XET-technology was utilized for enzymatic incorporation of a thiol moiety at one end of the xyloglucan backbone. This functionalized macromolecule was subsequently top-down grafted to gold, forming a thiol-bonded xyloglucan brushlike layer. The grafting was monitored in situ with QCM-D, and a significant difference in the adsorbed/grafted amount between unmodified xyloglucan and the thiol-functionalized polymer was observed. The grafted surface was demonstrated to be accessible to enzyme digestion using the plant endo-xyloglucanase TmNXG1. The nanotribological properties toward cellulose of the untreated crystal, brush-modified surface, and enzyme-exposed surfaces were compared with a view to understanding the role of xyloglucan in friction reduction. Friction coefficients obtained by the AFM colloidal probe technique using a cellulose functionalized probe on the xyloglucan brush showed an increase of a factor of 2 after the enzyme digestion, and this result is interpreted in terms of surface roughness. Finally, the brush is shown to exhibit binding to cellulose despite its highly oriented nature.

Comparison of different methods to calibrate torsional spring constant and photodetector for atomic force microscopy friction measurements in air and liquid.

A number of atomic force microscopy cantilevers have been exhaustively calibrated by a number of techniques to obtain both normal and frictional force constants to evaluate the relative accuracy of the different methods. These were of either direct or indirect character-the latter relies on cantilever resonant frequencies. The so-called Sader [Rev. Sci. Instrum. 70, 3967 (1999)] and Cleveland [Rev. Sci. Instrum. 64, 403 (1993)] techniques are compared for the normal force constant calibration and while agreement was good, a systematic difference was observed. For the torsional force constants, all the techniques displayed a certain scatter but the agreement was highly encouraging. By far the simplest technique is that of Sader, and it is suggested in view of this validation that this method should be generally adopted. The issue of the photodetector calibration is also addressed since this is necessary to obtain the cantilever twist from which the torsional force is calculated. Here a technique of obtaining the torsional photodetector sensitivity by combining the direct and indirect methods is proposed. Direct calibration measurements were conducted in liquid as well as air, and a conversion factor was obtained showing that quantitative friction measurements in liquid are equally feasible provided the correct calibration is performed.

Friction and forces between cellulose model surfaces: a comparison.

Four different cellulose model surfaces, and one silica surface, have been studied by means of atomic force microscopy (AFM). The normal interactions have been found to consist of a longer range double layer force with a short range steric interaction, the nature of which is extensively discussed. Both the surface charge and range of the steric force depend on the type of cellulose substrate used, as does the magnitude of the adhesion. Studies of friction reveal that surface roughness is the determining factor for the friction coefficient, with which it increases monotonically. The absolute value, however, is determined by the surface chemistry. All studied cellulose surfaces show similar behavior in response to xyloglucan addition.