Soft Biological Materials Research Articles

Soft biological materials and their impact on cell function.

Most organs and biological tissues are soft viscoelastic materials with elastic moduli ranging from on the order of 100 Pa for the brain to 100 000 Pa for soft cartilage. Biocompatible synthetic materials already have many applications, but combining chemical compatibility with physiologically appropriate mechanical properties will increase their potential for use both as implants and as substrates for tissue engineering. Understanding and controlling mechanical properties, specifically softness, is important for appropriate physiological function in numerous contexts. The mechanical properties of the substrate on which, or within which, cells are placed can have as large an impact as chemical stimuli on cell morphology, differentiation, motility, and commitment to live or die.

Spherical indentation load-relaxation of soft biological tissues

Elastic-viscoelastic correspondence was used to generate displacement–time solutions for spherical indentation testing of soft biological materials with time-dependent mechanical behavior. Boltzmann hereditary integral operators were used to determine solutions for indentation load-relaxation following a constant displacement rate ramp. A “ramp correction factor” approach was used for routine analysis of experimental load-relaxation data. Experimental load-relaxation tests were performed on rubber, as well as kidney tissue and costal cartilage, two hydrated soft biological tissues with vastly different mechanical responses. The experimental data were fit to the spherical indentation ramp-relaxation solutions to obtain values of short- and long-time shear modulus and of material time constants. The method is used to demonstrate linearly viscoelastic responses in rubber, level-independent indentation results for costal cartilage, and age-independent indentation results for kidney parenchymal tissue.

Polymer-Supported Membranes: Physical Models of Cell Surfaces

The functional modification of solid surfaces with plasma membrane models has been drawing increasing attention as a straightforward strategy to bridge soft biological materials and hard inorganic materials. Planar model membranes can be deposited either directly on solid substrates (solid-supported membranes), or on ultrathin polymer supports (polymer-supported membranes) that mimic the generic role of the extracellular matrix and the cell surface. The first part of this review provides an overview of advances in the fabrication of polymer-supported membranes. The middle section describes how such thin polymer interlayers can physically modulate the membrane–substrate contact. The last section introduces several methods to localize membranes and membrane proteins. Finally, some ideas are presented on combining supported membrane concepts with semiconductor technology toward applications in materials science.

On experimental testing methods for characterizing the mechanical properties of soft biological materials such as arterial tissues

On experimental testing methods for characterizing the mechanical properties of soft biological materials such as arterial tissues

102 ファンデルポール型自励発振方式原子間力顕微鏡(AFM)の開発 : 分岐制御を用いたカンチレバープローブの振幅制御

We propose a new control method of the micro cantilever probe in atomic force microscopy (AFM) for biologic imaging. The observation is concerned with operation in water or aqueous bufferin where the Q value of the cantilever is reduced. In this situation, it is very difficult to obtain the natural frequency of the micro cantilever probe. The utilization of self-excited oscillation is known as a method to overcome the difficulty. Furthermore, for the detection of the characteristics of soft biological material, the non-contact mode, that the probe does not contact to the material, is required for avoidance of local deformation and destruction of the material. In this study, for realizing the no contact mode of the self-excited micro cantilever probe, van der Pol type self-excited micro cantilever beam is designed by bifurcation control due to linear-plus-nonlinear feedback control.

Mechanical Characterization of Anisotropic Planar Biological Soft Tissues Using Large Indentation: A Computational Feasibility Study

Traditionally, the complex mechanical behavior of planar soft biological tissues is characterized by (multi)axial tensile testing. While uniaxial tests do not provide sufficient information for a full characterization of the material anisotropy, biaxial tensile tests are difficult to perform and tethering effects limit the analyses to a small central portion of the test sample. In both cases, determination of local mechanical properties is not trivial. Local mechanical characterization may be performed by indentation testing. Conventional indentation tests, however, often assume linear elastic and isotropic material properties, and therefore these tests are of limited use in characterizing the nonlinear, anisotropic material behavior typical for planar soft biological tissues. In this study, a spherical indentation experiment assuming large deformations is proposed. A finite element model of the aortic valve leaflet demonstrates that combining force and deformation gradient data, one single indentation test provides sufficient information to characterize the local material behavior. Parameter estimation is used to fit the computational model to simulated experimental data. The aortic valve leaflet is chosen as a typical example. However, the proposed method is expected to apply for the mechanical characterization of planar soft biological materials in general.

Experimental and numerical investigations of the deformation of soft materials under tangential loading

The surface ‘tensile test’, in which tangential loads are applied through surface mounted adhesive tapes, is a viable method for the assessment of mechanical properties of soft materials, particularly biological soft materials in vivo. In the present work the deformation pattern and force–displacement relationship in the surface tensile test were experimentally investigated using surface displacement analysis (SDA) and numerically simulated using finite element modelling. The experimental and FE results showed close agreement using silicone rubber as a model material. The force–displacement relationship was found to be dependent on the tape separations. SDA measurements and FE simulation showed that the displacement and strain fields were not uniform and the distribution pattern varies with tape separation. A combined experimental–numerical approach to inversely extract material properties using multiple tests with different length scales is proposed and assessed using a model material.

A simple cryogenic holder for tensile testing of soft biological tissues

To overcome the difficulty of gripping soft biological materials for tensile test, a simple inexpensive cryogenic holder was developed which allows rapid (3 min) preparation of samples. It is made of 6 parts, built in a bakelite cloth, which is an excellent thermal isolant, and is used with rectangular (8×10 −2×10 −2×10 −2 m) samples. The holder with the sample inside is completely immersed in liquid nitrogen for 50 s. This duration allows the freezing of the sample ends on a 10 −2 m length and gives a very flat freezing surface throughout the sample cross section. The 6×10 −2 m central part of the sample remained at ambient temperature. Two parts of the holder help maintain the sample until its ends are vertically gripped in the tensile machine thus avoiding any sample deformation during this step. No pressure was applied on the frozen part of the sample by grips of the tensile machine and this avoids breaks in this region. The sample is fixed by adhesion forces (>1 kN) between its frozen parts and 2 pieces of the holder. The procedure has been successfully tested with bovine and salmon muscle samples and results show tensile breaks randomly distributed in the unfrozen region of the samples. Particular attention has been paid to obtain a very flat freezing surface so that the axial strain is equal throughout the sample and therefore any strain-related mechanical parameters can be accurately determined. The dimensions of the holder can be easily modified to fit other sample geometries and can be used with other biological materials.

Special interfaces: military transformations

The objective of this paper is to review the mechanism of military transformations. Evidence is presented showing that these diffusionless transformations, which involve shear dominant deformation concomitant with interface motion in stiff engineering materials, occur by motion of disconnections along the interfaces. Disconnections are interfacial line-defects that exhibit both dislocation and step character. In the theoretical part of the review, topological arguments are used to identify the constraints on diffusionless motion of disconnections, and it is proposed that five criteria (one compositional, three crystallographic and one relating to the intrinsic mobility of disconnections) must be satisfied for feasible transformations. Subsequently, experimental observations in heterophase interfaces, namely, martensitic transformations, and homophase materials, i.e. deformation twinning and intervariant boundaries, are presented and interpreted in this framework. The generic nature of this mechanistic approach is further illustrated by a military transformation in a soft biological material, and a diffusive-displacive transformation in an intermetallic alloy where the compositional criterion is relaxed.

Ionic surfactant films imaged by atomic force microscopy

Forces acting on atomic force microscope (AFM) tips responsible for image formation are measured during scanning of films of ionic surfactant molecules adsorbed from aqueous solutions onto hydrophilic substrates. Near the critical micelle concentration mica substrate images show aggregate regions at the interface. Force versus distance measured curves show that patches form a thicker structure than the formed at partially covered regions, in agreement with the fact that at patches the adsorbates are perpendicularly oriented to the substrate plane. However, AFM topographic images registered at low scanning speed (5 μm/s) show that these patched regions appear as holes, forming inverted images. In AFM imaging of soft structures, as surfactants or biological material, inverted in contrast with images may be observed when there is a specific tip penetration through each scanned layer. This penetration is adjusted by changing the tip force set point, consequently different topographic profiles are obtained. The precise force set point to obtain the correct contrast in scanned images is obtained by the analysis of the force versus distance curves that show the normal to the scanned plane structure profile. Adsorption patterns as a function of time may be conveniently monitored and the adsorption rate may be determined.

High-Resolution Scanning Tunneling Microscopy of Fully Hydrated Ripple-Phase Bilayers

A modified freeze-fracture replication technique for use with the scanning tunneling microscope (STM) has provided a quantitative, high-resolution description of the waveform and amplitude of rippled bilayers in the P beta' phase of dimyristoylphosphatidylcholine (DMPC) in excess water. The ripples are uniaxial and asymmetrical, with a temperature-dependent amplitude of 2.4 nm near the chain melting temperature that decreases to zero at the chain crystallization temperature. The wavelength of 11 nm does not change with temperature. The observed ripple shape and the temperature-induced structural changes are not predicted by any current theory. Calibration and reproducibility of the STM/replica technique were tested with replicas of well-characterized bilayers of cadmium arachidate on mica that provide regular 5.5-nm steps. STM images were analyzed using a cross-correlation averaging program to eliminate the effects of noise and the finite size and shapes of the metal grains that make up the replica. The correlation averaging allowed us to develop a composite ripple profile averaged over hundreds of individual ripples measured on different samples with different STM tips. The STM/replica technique avoids many of the previous artifacts of biological STM imaging and can be used to examine a variety of periodic hydrated lipid and protein samples at a lateral resolution of about 1 nm and a vertical resolution of about 0.3 nm. This resolution is superior to conventional and tapping mode AFM to soft biological materials; the technique is substrate-free, and the conductive and chemically uniform replicas make image interpretation simple and direct.

An automatic micrometer for measuring soft electrically conductive materials

A device has been developed to determine the size of many soft biological material samples, which cannot be measured successfully with conventional micrometers because of distortion due to the force applied to the sample, It consists of a motor-driven, digital micrometer and control circuit, which is governed by the electrical conductivity of the material to be measured. The micrometer may be driven in an opening or closing mode by operating a three-position switch, but, when electrical contact is established with the sample surface, further closing is precluded.

Adhesive forces between ligand and receptor measured by AFM

Since its invention, the atomic force microscope (AFM) has been used to image a wide range of samples, including soft biological materials. Although various imaging modes have been developed in recent years, the images obtained by these techniques are primarily derived from the mechanical interactions between AFM tip and sample. An imaging mode based on the specific recognition of defined functional groups on the sample by receptor molecules attached to the AFM tip may prove to be beneficial, especially in complex specimens such as the cell membrane. A prerequisite towards this goal is the development of AFM tips functionalized with ligand-specific receptors. Here we report the fabrication of tips functionalized with either avidin or antibodies. These functionalized tips were characterized by force scan measurements on ligands cross-linked to agarose beads. The specificity of the observed adhesion between tip and sample was confirmed by specific blocking of the ligands or the receptors.

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm).

Attractive-mode imaging of biological materials with dynamic force microscopy

We have applied the non-contact dynamic force microscopy method to investigate soft biological materials such as hexagonally-packed intermediate layers, DNA, and tobacco mosaic virus under ambient conditions. This method, where a stiff cantilever is oscillated close to its resonance frequency with an amplitude of 0.3-1.5 nm above the sample, allows highly reliable investigation of soft organic matter with minimized normal and lateral forces between tip and sample. The vertical and lateral resolution are determined to be <1 AA and 1-2 nm, respectively, comparing favorably to established results from repulsive-mode scanning force microscopy experiments on adsorbate covered surfaces in liquids. The interaction forces are found to be attractive, dominated by damping mechanisms and attractive force gradients of capillary and van der Waals interactions.

Open-ended coaxial-line technique for the measurement of the microwave dielectric constant for low-loss solids and liquids

A system which enables fast and reliable measurements of the dielectric constant over continuous microwave frequency ranges for both solid and liquid low-loss materials is described. The main thrust of this work is the application of the open-ended coaxial-line probe technique, which has been used previously for soft biological materials, to low-loss solid samples. Using the instrumentation and procedure presented here, the dielectric constant for low-loss solids can be measured absolutely to ±2%–3% with routine care. The uncertainty can be reduced by about a factor of 2 by averaging several measurements. It is also smaller for liquid samples. This application features the use of relatively simple and readily available microwave components. Also, it is shown that a simple empirical relationship can be used to obtain the bulk dielectric constant from samples of a material in the form of thin slabs. The experimental results which are presented here for kapton, Teflon, Corning glass No. 0211, soda lime glass, magnesium oxide, sapphire, silicon, alumina, and lanthanum aluminate, as well as carbon tetrachloride, compare favorably with existing literature values.

Scan speed limit in atomic force microscopy

SUMMARYThe scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantilever k, its effective mass m, the damping constant D of the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measure k, k/m and D/m are described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μm/s.

The Challenge of Spectroscopy in the Microanalysis of Biological Surfaces

Abstract Chemical interactions between solids and their environments are initially characterized by the chemical composition of the surfaces. This is especially true for biochemical processes, such as ionic transport through biological membranes, the maintenance of mineral ion homeostasis which is critically dependent on the activity of the “bone membrane,” the biocompatibility of surgical implants which appears to be only a surface problem, etc. This is because biological surfaces have shown an increased interest for different fields of science. However, biological surfaces are heterogeneous and generally more complex than inorganic ones, and biological subsurfaces (internal surfaces) are not usually identical with the external surfaces. Taking into account that much of the functional significance of biological surfaces is related to the internal surfaces and these are normally hidden, we must expose them for analysis without introducing artifacts during the preparative procedures. As a result, the specimen preparation techniques are to a large extent the first limiting factor. Preparation of fully mineralized tissues such as teeth, bone, and kidney stones consisting of embedding in resin, sectioning, and fine polishing have been successfully accomplished. However, soft biological materials are prepared by cryogenic methods to preserve the original elemental distributions (but altering the tissue morphology) or by chemical fixation and water replacement by embedding media to preserve the morphology (but increasing diffusion and translocation of elements).

How SIMS microscopy can be used in medicine

The identification, localization and quantification of intracellular chemical elements is an area of scientific endeavour which has not ceased to develop over the past 30 years. Secondary Ion Mass Spectrometry (SIMS) microscopy is widely used for elemental localization problems in geochemistry, metallurgy and electronics. Although the first commercial instruments were available in 1968, biological applications have been gradual as investigators have systematically examined the potential source of artefacts inherent in the method and sought to develop strategies for the analysis of soft biological material with a lateral resolution equivalent to that of the light microscope. In 1992, the prospects offered by this technique are even more encouraging as prototypes of new ion probes appear capable of achieving the ultimate goal, namely the quantitative analysis of micron and submicron regions. The purpose of this review is to underline the requirements for biomedical applications of SIMS microscopy.Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue.

Atomic force microscopy for high-resolution imaging in cell biology

Historically, microscopy has been a tool of primary importance to the cell biologist. The scanning tunnelling microscope I started a revolution in microscopy that has led to the emergence of a family of scanning probe microscopes. These instru- ments pass a probe in close proximity to a surface, and collect spatially re- solved information about surface properties, such as tunnelling current, physical topography, ion conduc- tance or temperature 2. One of the most promising members of this family of instruments for biological applications is the atomic force microscope (AFM, also known as the scanning force microscope)3, 4. On hard samples the AFM can often resolve atoms, while on soft biological materials the lateral resol- ution is currently 1-50 nm. Its ability to image in fluids, including physio- logical buffers, and to gather dy- namic data points to a bright future