Synergy of Fiber Surface Chemistry and Flow: Multi-Phase Transcrystallization in Fiber-Reinforced Thermoplastics.

Fiber-reinforced polymer composites are largely employed for their improved strength with respect to unfilled matrices. Considering semi-crystalline materials under relevant processing conditions, the applied pressure and flow induce shear stresses at the fiber-polymer interface. These stresses may strongly enhance the nucleation ability of the fiber surface with respect to the quiescent case. It is thus possible to assume that the fiber features are no longer of importance and that crystallization is dominated by the effect of flow. However, by making use of an advanced experimental technique, i.e., polarization-modulated synchrotron infrared microspectroscopy (PM-SIRMS), we are able to show that the opposite is true for the industrially relevant case of isotactic polypropylene (iPP). With PM-SIRMS, the local chain orientation is measured with micron-size spatial resolution. This orientation can be related to the polymer nucleation density along the fiber surface. For various combinations of an iPP matrix and fiber, the degree of orientation in the cylindrical layer that develops during flow correlates well with the differences in nucleation density found in quiescent conditions. This result shows that the morphological development during processing of polymer composites is not solely determined by the flow field, nor by the nucleating ability of the fiber surface alone, but rather by a synergistic combination of the two. In addition, using finite element modeling, it is demonstrated that, under the experimentally applied flow conditions, the interphase structure formation is mostly dominated by the rheological characteristics of the material rather than perturbations in experimental conditions, such as shear rate, layer thickness, and temperature. This once again highlights the importance of matrix-filler interplay during flow and, thus, of material selection in the design of hybrid and lightweight composite technologies.

Response of Elementary Structural Transitions in Glassy Atactic Polystyrene to Temperature and Deformation.

The effects of temperature, pressure, and imposed strain on the structural transition pathways of glassy atactic polystyrene (aPS) are studied for a wide range of conditions. By employing an atomistic description of the system, we systematically explore its free energy landscape, emphasizing connections between local free energy minima. A triplet of two minima connected to each other via a first-order saddle point provides the full description of each elementary structural relaxation event. The basis of the analysis is the potential energy landscape (PEL), where efficient methods for finding saddle points and exploring transition pathways have been developed. We then translate the stationary points of the PEL to stationary points of the proper free energy landscape that obeys the macroscopically imposed constraints (either stress- or strain-controlled). By changing the temperature under isobaric conditions (i.e., Gibbs energy landscape), we probe the temperature dependence of the transition rates of the subglass relaxations of aPS, thus obtaining their activation energies by fitting to the Arrhenius equation. The imposition of different strain levels under isothermic conditions allows us to estimate the apparent activation volume of every elementary transition. Our findings are in good agreement with experimental observations for the same system, indicating that both length- and time-scales of the structural transitions of glassy aPS can be obtained by proper free energy minimization of atomistically detailed configurations.

Investigating the Morphology and Mechanics of Biogenic Hierarchical Materials at and below Micrometer Scale.

Investigating and understanding the intrinsic material properties of biogenic materials, which have evolved over millions of years into admirable structures with difficult to mimic hierarchical levels, holds the potential of replacing trial-and-error-based materials optimization in our efforts to make synthetic materials of similarly advanced complexity and properties. An excellent example is biogenic silica which is found in the exoskeleton of unicellular photosynthetic algae termed diatoms. Because of the complex micro- and nanostructures found in their exoskeleton, determining the intrinsic mechanical properties of biosilica in diatoms has only partly been accomplished. Here, a general method is presented in which a combination of in situ deformation tests inside an SEM with a realistic 3D model of the frustule of diatom Craspedostauros sp. (C. sp.) obtained by electron tomography, alongside finite element method (FEM) simulations, enables quantification of the Young's modulus (E = 2.3 ± 0.1 GPa) of this biogenic hierarchical silica. The workflow presented can be readily extended to other diatom species, biominerals, or even synthetic hierarchical materials.

In Situ Manipulation and Micromechanical Characterization of Diatom Frustule Constituents Using Focused Ion Beam Scanning Electron Microscopy.

Biocomposite structures are difficult to characterize by bulk approaches due to their morphological complexity and compositional heterogeneity. Therefore, a versatile method is required to assess, for example, the mechanical properties of geometrically simple parts of biocomposites at the relevant length scales. Here, it is demonstrated how a combination of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and micromanipulators can be used to isolate, transfer, and determine the mechanical properties of frustule constituents of diatom Thalassiosira pseudonana (T.p.). Specifically, two parts of the diatom frustule, girdle bands and valves, are separated by FIB milling and manipulated using a sharp tungsten tip without compromising their physical or chemical integrity. In situ mechanical studies on isolated girdle bands combined with Finite Element Method (FEM) simulations, enables the quantitative assessment of the Young's modulus of this biosilica; E = 40.0 GPa. In addition, the mechanical strength of isolated valves could be measured by transferring and mounting them on top of premilled holes in the sample support. This approach may be extended to any hierarchical biocomposite material, regardless of its chemical composition, to isolate, transfer, and investigate the mechanical properties of selected constituents or specific regions.

Structural Transitions in Glassy Atactic Polystyrene Using Transition-State Theory.

Transition pathways on the energy landscape of atactic polystyrene (aPS) glassy specimens are probed below its glass-transition temperature. Each of these transitions is considered an elementary structural relaxation event, whose corresponding rate constant is calculated by applying multidimensional transition-state theory. Initially, a wide spectrum of first-order saddle points surrounding local minima on the energy landscape is discovered by a stabilized hybrid eigenmode-following method. Then, (minimal-energy) "reaction paths" to the adjacent minima are constructed by a quadratic descent method. The heights of the free energy, the potential energy, and the entropy barriers are estimated for every connected triplet of transition state and minima. The resulting distribution of free energy barriers is asymmetric and extremely broad, extending to very high barrier heights (over 50 kBT); the corresponding distribution of rate constants extends over 30 orders of magnitude, with well-defined peaks at the time scales corresponding to the subglass relaxations of polystyrene. Analysis of the curvature along the reaction paths reveals a multitude of different rearrangement mechanisms; some of them bearing multiple distinct phases. Finally, connections to theoretical models of the glass phenomenology allows for the prediction, based on first-principles, of the "ideal" glass-transition temperature entering the Vogel-Fulcher-Tammann (VFT) equation describing the super-Arrhenius temperature dependence of glassy dynamics. Our predictions of the time scales of the subglass relaxations and the VFT temperature are in favorable agreement with available experimental literature data for systems of similar molecular weight under the same conditions.

A novel technique for the assessment of mechanical properties of vascular tissue.

Accurate estimation of mechanical properties of the different atherosclerotic plaque constituents is important in assessing plaque rupture risk. The aim of this study was to develop an experimental set-up to assess material properties of vascular tissue, while applying physiological loading and being able to capture heterogeneity. To do so, a ring-inflation experimental set-up was developed in which a transverse slice of an artery was loaded in the radial direction, while the displacement was estimated from images recorded by a high-speed video camera. The performance of the set-up was evaluated using seven rubber samples and validated with uniaxial tensile tests. For four healthy porcine carotid arteries, material properties were estimated using ultrasound strain imaging in whole-vessel-inflation experiments and compared to the properties estimated with the ring-inflation experiment. A 1D axisymmetric finite element model was used to estimate the material parameters from the measured pressures and diameters, using a neo-Hookean and Holzapfel-Gasser-Ogden material model for the rubber and porcine samples, respectively. Reproducible results were obtained with the ring-inflation experiment for both rubber and porcine samples. Similar mean stiffness values were found in the ring-inflation and tensile tests for the rubber samples as 202 kPa and 206 kPa, respectively. Comparable results were obtained in vessel-inflation experiments using ultrasound and the proposed ring-inflation experiment. This inflation set-up is suitable for the assessment of material properties of healthy vascular tissue in vitro. It could also be used as part of a method for the assessment of heterogeneous material properties, such as in atherosclerotic plaques.

A novel experimental setup for in situ optical and X-ray imaging of laser sintering of polymer particles.

We present a unique laser sintering setup that allows real time studies of the structural evolution during laser sintering of polymer particles. The device incorporates the main features of classical selective laser sintering machines for 3D printing of polymers and at the same time allows in situ visualization of the sintering dynamics with optical microscopy as well as X-ray scattering. A main feature of the setup is the fact that it provides local access to one particle-particle bridge during sintering. In addition, due to the small scale of the device and the specific laser arrangement process, parameters such as the temperature, laser energy, laser pulse duration, and spot size can be precisely controlled. The sample chamber provides heating up to 360 °C, which allows for sintering of commodity as well as high performance polymers. The latter parameters are controlled by the use of a visible light laser combined with an acousto-optic modulator for pulsing, which allows small and precise spot sizes and pulse times and pulse energies as low as 500 µs and 17 µJ. The macrostructural evolution of the particle bridge during sintering is followed via optical imaging at high speed and resolution. Placing the setup in high flux synchrotron radiation with a fast detector simultaneously allows in situ time-resolved X-ray characterizations. To demonstrate the capabilities of the device, we studied the laser sintering of two spherical PA12 particles. The setup provides crucial real-time information concerning the sintering dynamics as well as crystallization kinetics, which was not accessible up to now.

Physical Ageing of Polystyrene: Does Tacticity Play a Role?

The ageing kinetics of amorphous atactic (a-PS), isotactic (i-PS), and syndiotactic (s-PS) polystyrene were studied by means of flash-differential scanning calorimetry. The specimens were aged for up to 2 h at six different ageing temperatures: the optimum ageing temperature, that is, the temperature at which the enthalpy overshoot at the glass transition is maximal for the given elapsed time, and five ageing temperatures ranging from 20 to 80 K below the optimum ageing temperature. A logarithmic increase of the enthalpy overshoot with ageing time is observed for specimens at their optimum ageing temperatures. For temperatures significantly lower than the optimum, there is a range where the enthalpy overshoot is constant, but for higher temperatures (still below the optimum), a logarithmic increase is also observed. Moreover, the ageing kinetics appear to depend on tacticity, with s-PS and i-PS exhibiting the slowest and fastest ageing kinetics, respectively, and a-PS exhibiting ageing kinetics between these two extremes.

Laser sintering of polymer particle pairs studied by in situ visualization.

Merging of particle pairs during selective laser sintering (SLS) of polymers is vital in defining the final part properties. Depending on the sintering conditions, polymers can undergo full or partial sintering whereby incomplete sintering results in poor mechanical properties. At present, the underlying mechanisms and related conditions leading to various consolidation phenomena of polymer particles are not well understood. In the present work, a novel in-house developed experimental setup is used to perform laser sintering experiments on polystyrene (PS) particle doublets while performing in situ visualization of the sintering dynamics. From the recorded images, the evolution of the growth of the neck radius formed between both particles is analyzed as a function of time. Sintering conditions such as heating chamber temperature, laser pulse energy and duration, laser spot size and particle size are precisely controlled and systematically varied. A non-isothermal viscous sintering model is developed that allows qualitative prediction of the observed effects of the various parameters. It is shown that the sintering kinetics is determined by a complex interplay between the transient rheology caused by the finite relaxation times of the polymer and the time-dependent temperature profile which also affects the polymer viscosity. The combination of a full material characterization with sintering experiments under well-defined conditions has resulted in a general understanding of the effects of material and process parameters on laser sintering. Thereby a strong foundation is laid for the route towards rational design of laser sintering.

Insertion Process of Ceramic Nanoporous Microneedles by Means of a Novel Mechanical Applicator Design.

Arrays of microneedles (MNAs) are integrated in an out-of-plane fashion with a base plate and can serve as patches for the release of drugs and vaccines. We used soft-lithography and micromolding to manufacture ceramic nanoporous (np)MNAs. Failure modes of ceramic npMNAs are as yet poorly understood and the question remained: is our npMNA platform technology ready for microneedle (MN) assembly into patches? We investigated npMNAs by microindentation, yielding average crack fracture forces above the required insertion force for a single MN to penetrate human skin. We further developed a thumb pressure-actuated applicator-assisted npMNA insertion method, which enables anchoring of MNs in the skin by an adhesive in one handling step. Using a set of simple artificial skin models, we found a puncture efficiency of this insertion method a factor three times higher than by applying thumb pressure on the npMNA base plate directly. In addition, this new method facilitated zero MN-breakage due to a well-defined force distribution exerted onto the MNs and the closely surrounding area prior to bringing the adhesive into contact with the skin. Owing to the fact that such parameter space exists, we can conclude that npMNAs by soft lithography are a platform technology for MN assembly into a patch.

Six-minute walking test done in a hallway or on a treadmill: how close do the two methods agree?

BACKGROUND: The 6-min walking test (6-MWT) is probably the most widely used test to measure the functional capacity in cardiac rehabilitation. Although the American Thoracic Society recommends testing on a flat surface, treadmills are also used for testing. Therefore, we want to investigate the interchangeability of results of treadmill and hallway 6-MWT in a population of patients participating in a cardiac rehabilitation programme. DESIGN: Preexperimental design. SETTING: University hospital Department of Cardiology and Physiotherapy. PARTICIPANTS: Patients entering the cardiac rehabilitation programme of the Maastricht University Cardiology Department. MAIN OUTCOME MEASURE: Agreement in 6-min walking distance between the hallway and treadmill test results were calculated by taking the mean difference between the two methods and the 95% confidence interval of the difference and plotting this against the average of the two test results. A Bland and Altman plot was constructed, showing the mean difference and the 95% limits of agreement between the two methods. RESULTS: Sixty-nine patients participated in this study. Mean difference between walking on a treadmill and walking in a hallway was 9 m in favour of the hallway test. The 95% limits of agreement were±118 m. CONCLUSION: Results of the 6-MWT conducted in a hallway or on a treadmill are not interchangeable, because of large between-test variations in the distances walked by individual participants.

Genetic structure in African populations: implications for human demographic history.

The continent of Africa is the source of all anatomically modern humans that dispersed across the planet during the past 100,000 years. As such, African populations are characterized by high genetic diversity and low levels of linkage disequilibrium (LD) among loci, as compared to populations from other continents. African populations also possess a number of genetic adaptations that have evolved in response to the diverse climates, diets, geographic environments, and infectious agents that characterize the African continent. Recently, Tishkoff et al. (2009) performed a genome-wide analysis of substructure based on DNA from 2432 Africans from 121 geographically diverse populations. The authors analyzed patterns of variation at 1327 nuclear microsatellite and insertion/deletion markers and identified 14 ancestral population clusters that correlate well with self-described ethnicity and shared cultural or linguistic properties. The results suggest that African populations may have maintained a large and subdivided population structure throughout much of their evolutionary history. In this chapter, we synthesize recent work documenting evidence of African population structure and discuss the implications for inferences about evolutionary history in both African populations and anatomically modern humans as a whole.

Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases.

Small GTP-binding proteins of the Rho family (RhoA, Cdc42, Rac1) regulate the organisation and the turnover of the cell's cytoskeleton and adhesion structures. A significant function of these cellular structures is to translate and counterbalance forces applied to, or generated by, cells in order to maintain homeostasis and control cell movement. We therefore hypothesised that Rho-GTPases are directly involved in cellular gravity perception and may participate in the alterations induced in microgravity. To define an adequate cellular model allowing to investigate this issue, we have established stable cell lines constitutively expressing active forms of either RhoA, Cdc42, or Rac1. The three cell lines differ by morphology and by their ability to form filopodia, lamellipodia, and bundles of actin stress fibers. Overexpression of the active form of either RhoA, Cdc42, or Rac1 is compatible with cell viability and does not affect cell population doubling time. Thus, our series of mutant cells appear well suited to gain further knowledge on the molecular mechanisms of cellular gravity perception.

The in vitro influences of neurotensin on the motility characteristics of human U373 glioblastoma cells.

Astrocytic tumours are associated with dismal prognoses due to their pronounced ability to diffusely invade the brain parenchyma. Various neuropeptides, including gastrin, are able to modulate tumour astrocyte migration. While neurotensin has been shown to influence the proliferation of glioma cells and the migratory ability of a large set of other cell types, its role in glioma cell migration has never been investigated. Neurotensin-induced modifications to the motility features of human U373 glioblastoma cells therefore constitute the topic of the present study. We evidenced that three subtypes of neurotensin receptors (NTR1, NTR2 and NTR3) are expressed in U373 glioblastoma cells, at least as far as their mRNAs are concerned. Treating U373 tumour cells with 10 nM neurotensin markedly modified the morphological patterns of these cells and also profoundly altered the organization of their actin cytoskeletons. Pull-down assays revealed that neurotensin induced the activation in U373 cells of both Rac1 and Cdc42 but not RhoA. Scratch wound assays evidenced that neurotensin (0.1 and 10 nM) very significantly inhibited wound colonization by U373 cells cultured in the absence of serum. In addition, quantitative phase-contrast videomicroscopy analyses showed that neurotensin decreases the motility levels of U373 glioblastoma cells when these cells are cultured on plastic. In sharp contrast, neurotensin stimulates the motility of U373 cells when they are cultured on laminin, which is a pro-adhesive extracellular matrix component ubiquitously secreted by glioma cells. Our data thus strongly suggest that, in addition to gastrin, neurotensin is a neuropeptide capable of modulating tumour astrocyte migration into the brain parenchyma.

Effects of constitutively active GTPases on fibroblast behavior.

The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing.

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

The aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression.

Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts.

Mechanical tension governs fibroblast proliferation and survival and the homeostasis of the extracellular matrix to adapt its resistance to the mechanical requirements of the organs. To consolidate this view, we analysed the effect of tension release on the expression of molecules involved in the architecture and stabilisation of the collagen fibres, namely the procollagens type I and III, the amino- and carboxy-procollagen peptidases (N-pCP and C-pCP) and lysyl oxidase. Cells were cultured in conditions of high mechanical stress in monolayer on a collagen coat and under reduced tension by disruption of the cytoskeleton upon treatment with cytochalasin D in monolayer on a collagen coat or by integrin-mediated stress relaxation in a freely retracting collagen gel. The mRNAs were measured by quantitative RT-PCR monitored by simultaneous reverse-transcription and amplification of an original internal standard. Tension relaxation resulted in a decreased expression of the procollagens type I and III, of the two expressed forms of C-pCP, of the two forms of N-pCP and of lysyl oxidase. Type III collagen, known to control diameter of the fibres, was less down-regulated than type I collagen. Interestingly, the expression of the two alternatively spliced forms of the N-pCP was dissimilarly regulated. These data suggest that mechanical tension may modulate the stiffness of the extracellular matrix by controlling not only the level of expression of its fibrillar constituents but also that of the enzymes participating in their extracellular processing and mechanical stabilisation.

Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxy- and amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.