Philipp J. Thurner

Design and Modeling of a High-Speed AFM-Scanner

A new mechanical scanner design for a high-speed atomic force microscope (AFM) is presented and discussed in terms of modeling and control. The positioning range of this scanner is 13 mum in the X- and Y-directions and 4.3 mum in the vertical direction. The lowest resonance frequency of this scanner is above 22 kHz. This paper is focused on the vertical direction of the scanner, being the crucial axis of motion with the highest precision and bandwidth requirements for gentle imaging with the AFM. A second- and a fourth-order mathematical model of the scanner are derived that allow new insights into important design parameters. Proportional-integral (Pl)-feedback control of the high-speed scanner is discussed and the performance of the new AFM is demonstrated by imaging a calibration grating and a biological sample at 8 frames/s.

A new look at the pathogenesis of asthma

Asthma is an inflammatory disorder of the conducting airways that has strong association with allergic sensitization. The disease is characterized by a polarized Th-2 (T-helper-2)-type T-cell response, but in general targeting this component of the disease with selective therapies has been disappointing and most therapy still relies on bronchodilators and corticosteroids rather than treating underlying disease mechanisms. With the disappointing outcomes of targeting individual Th-2 cytokines or manipulating T-cells, the time has come to re-evaluate the direction of research in this disease. A case is made that asthma has its origins in the airways themselves involving defective structural and functional behaviour of the epithelium in relation to environmental insults. Specifically, a defect in barrier function and an impaired innate immune response to viral infection may provide the substrate upon which allergic sensitization takes place. Once sensitized, the repeated allergen exposure will lead to disease persistence. These mechanisms could also be used to explain airway wall remodelling and the susceptibility of the asthmatic lung to exacerbations provoked by respiratory viruses, air pollution episodes and exposure to biologically active allergens. Variable activation of this epithelial–mesenchymal trophic unit could also lead to the emergence of different asthma phenotypes and a more targeted approach to the treatment of these. It also raises the possibility of developing treatments that increase the lungs resistance to the inhaled environment rather than concentrating all efforts on trying to suppress inflammation once it has become established.

Osteopontin Deficiency Increases Bone Fragility but Preserves Bone Mass

The ability of bone to resist catastrophic failure is critically dependent upon the material properties of bone matrix, a composite of hydroxyapatite, collagen type I, and noncollagenous proteins. These properties include elastic modulus, hardness, and fracture toughness. Like other aspects of bone quality, matrix material properties are biologically-defined and can be disrupted in skeletal disease. While mineral and collagen have been investigated in greater detail, the contribution of noncollagenous proteins such as osteopontin to bone matrix material properties remains unclear. Several roles have been ascribed to osteopontin in bone, many of which have the potential to impact material properties. To elucidate the role of osteopontin in bone quality, we evaluated the structure, composition, and material properties of bone from osteopontin-deficient mice and wild-type littermates at several length scales. Most importantly, the results show that osteopontin deficiency causes a 30% decrease in fracture toughness, suggesting an important role for OPN in preventing crack propagation. This significant decline in fracture toughness is independent of changes in whole bone mass, structure, or matrix porosity. Using nanoindentation and quantitative backscattered electron imaging to evaluate osteopontin-deficient bone matrix at the micrometer level, we observed a significant reduction in elastic modulus and increased variability in calcium concentration. Matrix heterogeneity was also apparent at the ultrastructural level. In conclusion, we find that osteopontin is essential for the fracture toughness of bone, and reduced toughness in osteopontin-deficient bone may be related to the increased matrix heterogeneity observed at the micro-scale. By exploring the effects of osteopontin deficiency on bone matrix material properties, composition and organization, this study suggests that reduced fracture toughness is one mechanism by which loss of noncollagenous proteins contribute to bone fragility.

Epithelial mechanobiology, skin wound healing, and the stem cell niche

Skin wound healing is a vital process that is important for re-establishing the epithelial barrier following disease or injury. Aberrant or delayed skin wound healing increases the risk of infection, causes patient morbidity, and may lead to the formation of scar tissue. One of the most important events in wound healing is coverage of the wound with a new epithelial layer. This occurs when keratinocytes at the wound periphery divide and migrate to re-populate the wound bed. Many approaches are under investigation to promote and expedite this process, including the topical application of growth factors and the addition of autologous and allogeneic tissue or cell grafts. The mechanical environment of the wound site is also of fundamental importance for the rate and quality of wound healing. It is known that mechanical stress can influence wound healing by affecting the behaviour of cells within the dermis, but it remains unclear how mechanical forces affect the healing epidermis. Tensile forces are known to affect the behaviour of cells within epithelia, however, and the material properties of extracellular matrices, such as substrate stiffness, have been shown to affect the morphology, proliferation, differentiation and migration of many different cell types. In this review we will introduce the structure of the skin and the process of wound healing. We will then discuss the evidence for the effect of tissue mechanics in re-epithelialisation and, in particular, on stem cell behaviour in the wound microenvironment and in intact skin. We will discuss how the elasticity, mechanical heterogeneity and topography of the wound extracellular matrix impact the rate and quality of wound healing, and how we may exploit this knowledge to expedite wound healing and mitigate scarring.

Non-destructive three-dimensional evaluation of a polymer sponge by micro-tomography using synchrotron radiation

X-ray micro-tomography, a non-destructive technique is used to uncover the complex 3-D micro-architecture of a degradable polymer sponge designed for bone augmentation. The measurements performed at HASYLAB at DESY are based on a synchrotron radiation source resulting in a spatial resolution of about 5.4 microm. In the present communication we report the quantitative analysis of the porosity and of the pore architecture. First, we elucidate that synchrotron radiation at the photon energy of 9 keV has an appropriate cross section for this low-weight material. Modifications in sponge micro-architecture during measurement are not detected. Second, the treatment of the data, an amount of 2.5 Gbyte to generate binary data is described. We compare the 3-D with the 2-D analysis in a quantitative manner. The obtained values for the mean distance to material within the sponge calculated from 2-D and 3-D data of the whole tomogram differ significantly: 12.5 microm for 3-D and 17.6 microm for 2-D analysis. If the pores exhibit a spherical shape as frequently found, the derived mean pore diameter, however, is overestimated only by 6% in the 2-D image analysis with respect to the 3-D evaluation. This approach can be applied to different porous biomaterials and composites even in a hydrated state close to physiological conditions, where any surface preparation artifact is avoided.

Atomic force microscopy and indentation force measurement of bone

This review is summarizing the results obtained from atomic force microscopy (AFM) and nanoindentation experiments to date. The combination of both techniques is especially powerful. It allows to carefully choose indentation locations as well as the post-hoc analysis of the created indents, and hence the possibility to assess the properties of microstructural elements of bonessue. In addition, AFM has improved our understanding of bone ultrastructure and force spectroscopy experiments have led to the discovery of a molecular self-healing effect of bone that may be based on a small fraction of unstructured proteins. Nanoindentation measurements on bone, pose inherent problems since bone is an anisotropic solid showing elastic, viscoelastic, and time-dependent plastic behavior. Hence, derived parameters such as elastic modulus and hardness are to some extent dependent on measurement protocols. However, the development of extensions to the Oliver-Pharr method, being the most widely used analysis method, as well as novel dynamic testing techniques could improve the situation. Nanoindentation is widely used to study bone tissue and some important principal findings have been reported to date. These are presented here together with specific results from nanoindentation experiments of human and animal bones and tables are presented collating the data that can be found in the literature to date.

Effect of Ca2+ ions on the adhesion and mechanical properties of adsorbed layers of human osteopontin.

Using an atomic force microscope and a surface force apparatus, we measured the surface coverage, adhesion, and mechanical properties of layers of osteopontin (OPN), a phosphoprotein of the human bones, adsorbed on mica. OPN is believed to connect mineralized collagen fibrils of the bone in a matrix that dissipates energy, reducing the risk of fractures. Atomic force microscopy normal force measurements showed large adhesion and energy dissipation upon retraction of the tip, which were due to the breaking of the many OPN-OPN and OPN-mica bonds formed during tip-sample contact. The dissipated energy increased in the presence of Ca(2+) ions due to the formation of additional OPN-OPN and OPN-mica salt bridges between negative charges. The forces measured by surface force apparatus between two macroscopic mica surfaces were mainly repulsive and became hysteretic only in the presence of Ca(2+): adsorbed layers underwent an irreversible compaction during compression due to the formation of long-lived calcium salt bridges. This provides an energy storage mechanism, which is complementary to energy dissipation and may be equally relevant to bone recovery after yield. The prevalence of one mechanism or the other appears to depend on the confinement geometry, adsorption protocol, and loading-unloading rates.

Nondestructive three-dimensional evaluation of biocompatible materials by microtomography using synchrotron radiation

Microtomography based on synchrotron radiation sources is a unique technique for the 3D characterization of different materials with a spatial resolution down to about 1 micrometers . The interface between implant materials (metals, ceramics and polymers) and biological matter is nondestructively accessible, i.e. without preparation artifacts. Since the materials exhibit different x-ray absorption, it can become impossible to visualize implant material and tissue, simultaneously. Here, we show that coating of polymer implants, which are invisible in bone tissue, does not only improve the interfacial properties but also allows the imaging of the interface in detail. Titanium implants, on the other hand, absorb the x-rays stronger than bone tissue. The difference, however, is small enough to quantify the bone formation near interface. Another advantage of microtomography with respect to classical histology is the capability to examine samples in a hydrated state. We demonstrate that ceramic hollow spheres can be imaged before sintering and fibroblasts marked by OsO4 are visible on polymer textiles. Consequently, scaffolds of different materials designed for tissue engineering and implant coatings can be optimized on the basis of the tomograms.

Extracellular DNA Impedes the Transport of Vancomycin in Staphylococcus epidermidis Biofilms Preexposed to Subinhibitory Concentrations of Vancomycin

ABSTRACT Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d-Ala-d-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

Design and modeling of a high-speed scanner for atomic force microscopy

A new scanner design for a high-speed atomic force microscope (AFM) is presented and discussed in terms of modeling and control. The lowest resonance frequency of this scanner is above 22 kHz. The X and Y scan ranges are 13 micrometers and the Z range is 4.3 micrometers. The focus of this contribution is on the vertical positioning direction of the scanner, being the crucial axis of motion with the highest bandwidth and precision requirements for gentle imaging with the atomic force microscope. A mathematical model of the scanner dynamics is presented that will enable more accurate topography measurements with the high-speed AFM system