A new method for extracting quantitative data from amplitude modulation dynamic force-distance measurements is developed. The method is based on the harmonic oscillator model of vibrating atomic force microscope cantilevers, and is capable of extracting both the conservative and dissipative parts of the tip-sample interaction from a measurement of oscillation amplitude and phase as a function of distance. Numerical simulations are used to demonstrate the validity of the method. Further proof of the accuracy of this method is provided by a measurement of electrostatic forces between an AFM tip and a graphite sample.

The UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) Some rights reserved. For more information, please see the item record link above. TitleNano-textured self-assembled aligned collagen hydrogels promote directional neurite guidance and overcome inhibition by myelin associated glycoprotein The development of nerve guidance conduits is constantly evolving as the need arises for therapies for spinal cord injury. In addition to providing a path for regrowing axons to reconnect with their appropriate targets, the structural and biochemical cues provided by these conduits should be permissive for directional neurite outgrowth and be protective against inhibition in the vicinity of the injury site. Here, we adapted the use of iso-electric focusing to drive the alignment of supramolecular fibrils into self-assembled collagen hydrogels ($300 mm diameter), and tested those hydrogels for the ability to direct and enhance the migration of neurites. Structural characterization revealed anisotropic alignment of nanofibrillar aggregates ($20 nm diameter), arranged in micron-scale bundles ($1 to 2 mm diameter) similar to the hierarchical size scales observed in native tissues. Neurite outgrowth extended bidirectionally along the axes of aligned hydrogels. Furthermore, it was shown that, as opposed to poly-D-lysine, neurite outgrowth on aligned hydrogels is not inhibited in the presence of myelin-associated glycoprotein (p > 0.05). These results highlight for the first time a structural and biochemical role for iso-electrically aligned collagen hydrogels in controlling neuronal growth, and indicate that the short-term signaling associated with these hydrogels can be used in adjunct therapy following injury to the spinal cord.

a b s t r a c tMuscle contractions begin in early embryonic life, generating forces that regulate the correct formation of the skeleton. In this paper we test the hypothesis that the biophysical stimulation generated by muscle forces may be a causative factor for the changes in shape of the knee joint as it grows. We do this by predicting the spatial and temporal patterns of biophysical stimuli, where cell proliferation and rudiment shape changes occur within the emerging tissues of the joint over time. We used optical projection tomography (OPT) to create anatomically accurate finite element models of the embryonic knee at three time points (stages) of development. OPT was also used to locate muscle attachment sites and AFM was used to determine material properties. An association was found between the emergence of joint shape, cell proliferation and the pattern of biophysical stimuli generated by embryonic muscle contractions. Elevated rates of growth and cell proliferation in the medial condyle were found to co-localise with elevated patterns of biophysical stimuli including maximum principal stresses and fluid flow, throughout the time period studied, indicating that cartilage growth and chondrocyte proliferation in the epiphysis is potentially related to local patterns of biophysical stimuli. The development of the patella and articular cartilages, which is known to be affected by in ovo immobilisation, could be contributed to by specific patterns of fluid flow, pore pressure and stress in the joint interzone. This suggests that both cartilage growth and tissue differentiation in the embryonic joint is regulated by specific patterns of biophysical stimuli and that these stimuli are needed for the correct development of the joint.

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A compact, two-stage nanomanipulator was designed and built for use inside a scanning electron microscope. It consists of a fine stage employing piezostacks that provide a 15 microm range in three dimensions and a coarse stage based on commercially available stick-slip motors. Besides the fabrication of enhanced probes for scanning probe microscopy and the enhancement of electron field emitters, other novel manipulation processes were developed, such as locating, picking up, and positioning small nanostructures with an accuracy of approximately 10 nm. In combination with in situ I-V experiments, welding, and etching, this results in a multipurpose nanofactory, enabling a new range of experiments.

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as “improved SP-D” (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans.

Atomic force microscopy (AFM) is widely used in the study of both morphology and mechanical properties of living cells under physiologically relevant conditions. However, quantitative experiments on timescales of minutes to hours are generally limited by thermal drift in the instrument, particularly in the vertical (z) direction. In addition, we demonstrate the necessity to remove all air-liquid interfaces within the system for measurements in liquid environments, which may otherwise result in perturbations in the measured deflection. These effects severely limit the use of AFM as a practical tool for the study of long-term cell behavior, where precise knowledge of the tip-sample distance is a crucial requirement. Here we present a readily implementable, cost effective method of minimizing z-drift and liquid instabilities by utilizing active temperature control combined with a customized fluid cell system. Long-term whole cell mechanical measurements were performed using this stabilized AFM by attaching a large sphere to a cantilever in order to approximate a parallel plate system. An extensive examination of the effects of sphere attachment on AFM data is presented. Profiling of cantilever bending during substrate indentation revealed that the optical lever assumption of free ended cantilevering is inappropriate when sphere constraining occurs, which applies an additional torque to the cantilevers “free” end. Here we present the steps required to accurately determine force-indentation measurements for such a scenario. Combining these readily implementable modifications, we demonstrate the ability to investigate long-term whole cell mechanics by performing strain controlled cyclic deformation of single osteoblasts.