The environmental scanning electron microscope (ESEM) offers a new approach for the examination of biological samples at rather high resolution. It permits samples to be imaged without extensive specimen preparation, and specifically requires neither drying nor coating. This implies that organic samples can be looked at in their native state, without the danger of artefacts being introduced. A few specific examples both of static imaging, and also images obtained during dynamic mechanical testing are given in this article to illustrate the power of the technique.

Most of the inorganic biomineralized materials are deposited on an organic matrix that controls the orientation and structure of the crystals. It is thought that chemical groups at the surface of the matrix may act as a template for the nucleation and growth of the mineral. A x-ray diffraction study of the texturization development of the bivalve mollusk shells is presented; specifically, the mussel Ischadium recurvum (Rafinesque, 1820), in different growing stages. The x-ray reflections show a preferred orientation that changes as the mollusk grows, and at the final stages only two crystallographic planes prevail.

Cell movement, differentiation and metabolic function must be controlled in precise ways to produce both regenerated tissues such as bone and functional tissue equivalents such as immuno-isolated islet cells. Close examination of extracellular matrix (ECM) reveals structures on the micron and nanometer scale that are shown to influence these factors and therefore we hypothesize that cells will move based on topographical cues in the scaffold. We have engineered siloxane elastomer surfaces that mimic the ECM by combining micron and nanometer scale topographic features. Micron scale pillars and ridges ranging in height from 1.5 to 5 microns and separated by 5, 10 and 20 microns were fabricated in a silicon wafer using micro- processing techniques and replicated in polydimethylsiloxane (PDMS) elastomer. Nanometer scale pillars, ridges and more complex shapes ranging in height from 12 to 300 nanometers were superimposed on the micron scale features using nanolithography. This was achieved by using a tapping mode tip in the atomic force microscope (AFM) to plastically deform the substrate surface. The AFM enabled nano-features to be placed on sloped surfaces and added directly to the PDMS elastomer surface. Surface topography was examined using scanning electron microscopy, atomic force microscopy and white light interference profilometry to verify surface modifications and fidelity of the replication process. Results indicate that it is possible to create spatially engineered surface textures from 10-5 m to 10-8 m in size, in specified patterns, by using a combination of microprocessing and nanolithography techniques. As better understanding of ECM function and design is gained, the processing methods outlined here will assist in fabricating tissue engineering scaffolds optimized at the nanometer and micron scale.

Disease processes are often associated with changes in tissue composition. For example, in atherosclerosis lipid and calcification are often found in the artery wall, whereas in healthy arteries the tissue microstructure is dominated by highly organized collagen. Such variations in composition likely result in changes in the material properties of the tissue. However, this relationship has not been fully investigated in atherosclerotic vessels. Using a combination of nanoindentation and spectroscopic techniques, our goal was to assess how changes in tissue composition affect the tissue's mechanical properties. Fourier Transform Infrared Spectroscopy (FTIR) was used to assess the biochemical composition of the tissue samples, such as the lipid and calcium content of fibrous tissues in diseased arteries. Nanoindentation was used to measure the local mechanical properties of the same tissue samples. This information was then correlated by position in the sample to assess the contributions of different constituents to the overall structure-property relations of these tissues.

The outstanding lubricating properties of synovial fluid, found in freely moving mammalian joints, may be due to intermolecular associations between hyaluronic acid, an anionic polysaccharide, and the plasma proteins. A synovial fluid model comprised of hyaluronic acid and the plasma proteins albumin and γ-globulins, was constructed. Rheological measurements reveal a pronounced viscoelasticity with a strong shear history dependence for the synovial fluid model and the plasma protein solutions at low shear rates. The addition of the anti-inflammatory drug D-Penicillamine to the solution alters the rheology of the synovial fluid model. We present two ideas about the structural features of synovial fluid that may explain this viscoelasticity and suggest further experimental techniques that can be used to test these ideas.

The potential of photothermal radiometry for subsurface defect imaging in shells of Crassostrea virginica is explored. It is shown that using this technique in the modality of modulated frequency, it is possible to obtain photothermal images in the case of small thermal diffusion lengths. By controlling the modulation frequency, it is shown that the technique can be useful in the study of buried defects in the shell. The application of this technique in monitoring living organisms is discussed.

A main limitation of biomedical devices is the inability to start, stop, and control cell growth making it crucial to develop biomaterial surfaces that induce a desired cellular response. Micropatterns of ridges and pillars were created in a siloxane elastomer (Dow Corning) by casting against epoxy replicates of a micromachined silicon wafer. Silicone oils were incorporated to determine the change in modulus and surface energy caused by these additives. SEM and white light interference profilometry verified that the micropatterning process produced high fidelity, low defect micropatterns. Mechanical analysis indicated that varying the viscosity, weight percent and functionality of the added silicone oil could change the elastic modulus by over an order of magnitude (0.1-2.3 MPa). As a self-wetting resin, silicone oils migrate to the surface, hence changing the surface properties from the bulk. Both topographical and chemical features define the surface energy, which in combination with elastic modulus, dictate biological activity. The results imply that the morphology, mechanical properties and surface energy of the siloxane elastomer can be modified to elicit a specific cell response as a function of engineered topographical and chemical functionalization.

Diacetylenic phospholipid dispersions in water produce tubules (500 nm diameter) and helices from their initial vesicular morphology as a function of temperature and concentration. A binary mixture consisting of diacetylenic phospholipid, 1, 2 bis (tricosa-10, 12-diynoyl)-sn-glycero-3-phosphocholine and a short chain phospholipid, 1, 2-dinonanoyl -sn-glycero-3-phosphocholine was studied to explore the morphological transformation of lipids into tubules to develop an approach to control and produce tubules of different diameters. Circular dichroic spectra not only indicated the chiral nature of these tubules, but also provided distinct spectral signatures differentiating micro- and nanotubules. The effects of temperature and lipid concentration on the formation and stability of tubules were also explored. An equimolar lipid mixture provided structures with uniform morphology, which were stable for several hours up to 36 °C. The thermal stability of nanotubules makes them an attractive candidate for many practical applications including controlled release technology.

Zebrafish (Danio rerio), the only vertebrate organism now amenable to large scale of “saturation genetic screens”, provides a single opportunity to investigate the complex gene mechanisms for normally vertebrate-specific processes. Studies of bone formation and bone diseases utilizing zebrafish system are therefore of interest. In the present study, microstructure and mechanical properties of skeletal bone in gene-mutated Stöpsel dtl28d (stp/stp) and wild-type zebrafish were investigated using transmission electron microscopy (TEM) and an atomic force microscopy (AFM)-based nanoindenter. Abnormal collagen matrix and minerals were observed in stp/stp bone comparing to the wild-type control. Nanoindentation measurement disclosed that there was a significant increase of elastic modulus in stp/stp bone compared to that of the control. AFM examinations of the residual indenter impressions indicate that the bone of zebrafish becomes more brittle after stp gene-mutation. This observation of the alteration of bone collagen, minerals and mechanical behavior by gene-mutation in zebrafish system is of scientific and clinical importance in many fields, such as bone fracture and brittleness in human heritable disorders and materials fabrication of bone tissue engineering via gene control.

We report a study on cellular traction forces and morphology on fibronectin (FN)- and Arg-Gly-Asp (RGD) modified substrata. We have focused on fibronectin- and RGD- modified substrata because RGD is a primary cell-binding site on fibronectin. In this study we report the traction stresses exerted by NIH (National Institutes of Health) 3T3 fibroblasts on model polyacrylamide hydrogel substrates using the elastic substrata technique. At equal values of input concentration of fibronectin and RGD we find that the values forprojected cell area are similar. However, a significant difference is observed in the traction forces exerted by fibroblasts on fibronectin- compared to RGD-modified surfaces. At equal values of input concentration, the average force exerted by NIH 3T3 fibroblasts is approximately ten fold higher on fibronectin-modified surfaces in comparison to RGD-modified surfaces.

Multiparametric optical analysis (MOA) can provide further progress in characterization and on-line control of biological and other disperse systems with nanoparticles as the disperse phase in various dispersive media. MOA includes the nondestructive simultaneous testing of disperse systems by different compatible optical methods such as refractometry, absorption, fluorescence, light scattering (integral and differential, static and dynamic, unpolarized and polarized). For the solution of inverse optical problems various techniques can be applied. By optical methods it is possible to determine parameters of disperse system's state (mean equivalent diameter and number of particles, mean refractive index and mass of disperse phase, number and mass distributions) and parameters of particle's structure. This paper is devoted to characterization of biological particles that can be approximated as bilayered spheres (blood-substitutes, liposomes, etc.) and to polarization measurements of disperse systems (viruses, bacterial cells, etc.) which can help to develop on-line control of environment.