Evaluating the viscoelastic properties of biological tissues in a new way

In this paper, a new method for evaluating the viscoelastic properties of biological tissues such as tendons and ligaments is presented. This method obtains the complex modulus of these tissues to characterize their viscoelastic properties. With this method, the stresses and strains measured in time are first transformed (using FFT), and the complex modulus is then obtained. The complex modulus contains sufficient information about the viscoelastic characteristics of the biological tissues. With this method, the mechanical properties of biological tissues can be measured without making apriori assumptions regarding their structures, and the measurements can be made in real time.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Nanomechanical Behaviours of Cuticle of Three Kinds of Beetle

The surface materials and structures of insect cuticle can provide useful information for designing anti-adhesion components material. Quantitative measurement of mechanical properties of insect cuticle will help to develop biomimetic materials suitable for industrial products. In this work, the mechanical properties, such as the reduced modulus and hardness in nano-scale, of the cuticle of beetle Geotrupes stercorarius Linnaeus, Copris ochus Motschulsky and Holotrichia sichotana Brenske, were investigated by using a nanoindenter. It was found that the reduce modulus and hardness of these three beetles are different. The main cause of the difference of the mechanical properties is probably due to their different living circumstance, lifestyle and different functions of segments.     

Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device

Knowledge of mechanical properties and failure mechanisms of biofilms is needed to determine how biofilms react on mechanical stress. Methods currently available cannot be used to determine mechanical properties of biofilms on a small scale with high accuracy. A novel microindentation apparatus in combination with a confocal microscope was used to determine the viscoelastic properties of Streptococcus mutans biofilms. The apparatus comprises a small glass indenter and a highly sensitive force transducer. It was shown that the present biofilm, grown under still conditions, behaves as a viscoelastic solid with a storage modulus of 1-8 kPa and a loss modulus of 5-10 kPa at a strain of 10%. Biofilm failure was investigated visually through a confocal microscope by dragging the indenter through the biofilm. It was shown that the tensile strength of the biofilm is predominantly determined by the tensile strength of the extracellular polysaccharide matrix. The combination of microindentation and confocal microscopy is a promising technique to determine and characterize the mechanical properties of soft materials in various fields of microbiology.     

Elastic contributions dominate the viscoelastic properties of sputum from cystic fibrosis patients

Sputum samples from cystic fibrosis (CF) patients were investigated by oscillatory, creep and steady shear rheological techniques over a range of time scales from 10(-3) to 10(6) s. The viscoelastic changes obtained by mixing sputa with the actin-filament-severing protein gelsolin and with the thiol-reducing agent dithiothreitol (DTT) were also investigated. At small strains sputum behaves like a viscoelastic solid rather than a liquid. A nearly constant steady shear viscosity at low shear rates is only observed after long shearing times which cause irreversible changes in the samples. Creep-recovery tests confirm that sputa exhibit viscoelastic properties, with a significant elastic recovery. The results suggest that measurements of elastic moduli, rather than viscosities are more closely related to the mechanical properties of sputum in situ. Severing of actin filaments lowers the elastic modulus by 30-40%, but maintains viscoelastic integrity, while reduction of thiols in the glycoproteins nearly completely fluidizes the samples.     

The importance of leaf cuticle for carbon economy and mechanical strength

Cuticle thickness of leaves varies >?100 times across species, yet its dry mass cost and ecological benefits are poorly understood. It has been repeatedly demonstrated that thicker cuticle is not superior as a water barrier, implying that other functions must be important. Here, we measured the mechanical properties, dry mass and density of isolated cuticle from 13 evergreen woody species of Australian forests. Summed adaxial and abaxial cuticle membrane mass per unit leaf area (CMA) varied from 2.95 to 27.4?g m(-2) across species, and accounted for 6.7-24% of lamina dry mass. Density of cuticle varied only from 1.04 to 1.24?g?cm(-3) ; thus variation in CMA was mostly due to variation in cuticle thickness. Thicker cuticle was more resistant to tearing. Tensile strength and modulus of elasticity of cuticle were much higher than those of leaf laminas, with significant differences between adaxial and abaxial cuticles. While cuticle membranes were thin, they could account for a significant fraction of leaf dry mass due to their high density. The substantial cost of thicker cuticle is probably offset by increased mechanical resistance which might confer longer leaf lifespans among evergreen species.     

Solvent effects on the viscoelastic behavior of porcine submaxillary mucin

Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1–5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G′, is much larger than the loss modulus, G″, at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G″ > G′ at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G′ ≈ G″ and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.     

Softness, strength and self-repair in intermediate filament networks

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.     

Viscoelastic properties of single attached cells under compression

The viscoelastic properties of single, attached C2C12 myoblasts were measured using a recently developed cell loading device. The device allows global compression of an attached cell, while simultaneously measuring the associated forces. The viscoelastic properties were examined by performing a series of dynamic experiments over two frequency decades (0.1-10 Hz) and at a range of axial strains (approximately 10-40%). Confocal laser scanning microscopy was used to visualize the cell during these experiments. To analyze the experimentally obtained force-deformation curves, a nonlinear viscoelastic model was developed. The nonlinear viscoelastic model was able to describe the complete series of dynamic experiments using only a single set of parameters, yielding an elastic modulus of 2120 +/- 900 Pa for the elastic spring, an elastic modulus of 1960 +/- 1350 for the nonlinear spring, and a relaxation time constant of 0.3 +/- 0.12 s. To our knowledge, it is the first time that the global viscoelastic properties of attached cells have been quantified over such a wide range of strains. Furthermore, the experiments were performed under optimal environmental conditions and the results are, therefore, believed to reflect the viscoelastic mechanical behavior of cells, such as would be present in vivo.     

Compressive properties of trabecular bone in the distal femur

Early loosening and implant migration are two problems that lead to failures in cementless (press-fit) femoral knee components of total knee replacements. To begin to address these early failures, this study determined the anterior-posterior mechanical properties from four locations in the human distal femur. Thirty-three cylindrical specimens were removed perpendicular to the press-fit surface after the surgical cuts on 10 human cadaveric femurs (age 71.5+/-14.2 years) had been made. Compression testing was performed that utilized methods to reduce the effects of end-artifacts. The bone mineral apparent density (BMAD), apparent modulus of elasticity, yield and ultimate stress, and yield and ultimate strain were measured for 28 cylindrical specimens. The apparent modulus, yield and ultimate stress, and yield and ultimate strain each significantly differed (p<0.05) in the superior and inferior locations. Linear and power law relationships between superior and inferior mechanical properties and BMAD were determined. The inferior apparent modulus and stresses were higher than those in the superior locations. These results show that the press-fit fixation characteristics of the femoral knee component differ on the anterior shield and posterior condyles. This information will be useful in the assignment of mechanical properties in finite element models for further investigations of femoral knee components. The property-density relations also have applications for implant design and preoperative assessment of bone strength using clinically available tools.     

A three-dimensional viscoelastic model for cell deformation with experimental verification

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

In vivo liver tissue mechanical properties by Transient Elastography: comparison with Dynamic Mechanical Analysis

Understanding the mechanical properties of human liver is one of the most critical aspects of its numerical modeling for medical applications or impact biomechanics. Generally, model constitutive laws come from in vitro data. However, the elastic properties of liver may change significantly after death and with time. Furthermore, in vitro liver elastic properties reported in the literature have often not been compared quantitatively with in vivo liver mechanical properties on the same organ. In this study, both steps are investigated on porcine liver. The elastic property of the porcine liver, given by the shear modulus G, was measured by both Transient Elastography (TE) and Dynamic Mechanical Analysis (DMA). Shear modulus measurements were realized on in vivo and in vitro liver to compare the TE and DMA methods and to study the influence of testing conditions on the liver viscoelastic properties. In vitro results show that elastic properties obtained by TE and DMA are in agreement. Liver tissue in the frequency range from 0.1 to 4 Hz can be modeled by a two-mode relaxation model. Furthermore, results show that the liver is homogeneous, isotropic and more elastic than viscous. Finally, it is shown in this study that viscoelastic properties obtained by TE and DMA change significantly with post mortem time and with the boundary conditions.     

Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone

Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference.The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30 s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.     

Influence of experimental protocols on the mechanical properties of the intervertebral disc in unconfined compression

The lack of standardization in experimental protocols for unconfined compression tests of intervertebral discs (IVD) tissues is a major issue in the quantification of their mechanical properties. Our hypothesis is that the experimental protocols influence the mechanical properties of both annulus fibrosus and nucleus pulposus. IVD extracted from bovine tails were tested in unconfined compression stress-relaxation experiments according to six different protocols, where for each protocol, the initial swelling of the samples and the applied preload were different. The Young's modulus was calculated from a viscoelastic model, and the permeability from a linear biphasic poroviscoelastic model. Important differences were observed in the prediction of the mechanical properties of the IVD according to the initial experimental conditions, in agreement with our hypothesis. The protocol including an initial swelling, a 5% strain preload, and a 5% strain ramp is the most relevant protocol to test the annulus fibrosus in unconfined compression, and provides a permeability of 5.0 ± 4.2e(-14)m(4)/N[middle dot]s and a Young's modulus of 7.6 ± 4.7 kPa. The protocol with semi confined swelling and a 5% strain ramp is the most relevant protocol for the nucleus pulposus and provides a permeability of 10.7 ± 3.1 e(-14)m(4)/N[middle dot]s and a Young's modulus of 6.0 ± 2.5 kPa.     

Viscoelastic properties of isolated collagen fibrils

Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests.     

Towards a reliable characterisation of the mechanical behaviour of brain tissue: The effects of post-mortem time and sample preparation

Since the early seventies, the material properties of brain tissue have been studied using a variety of testing techniques. However, data reported in literature show large discrepancies even in the linear viscoelastic regime. In the current study, the effect of the sample preparation procedure and of post-mortem time on the mechanical response of porcine brain tissue is examined. Samples from the thalamus region were prepared with different techniques and were tested for different loading histories. Each sample was tested in oscillatory shear tests (1% strain amplitude, 1-10 Hz frequencies) followed by sequences of 5% strain loading-unloading cycles. The stress response to the loading-unloading cycles showed a clear dependency on post-mortem time, becoming more stiff with increasing time. This dependency was affected by the mechanical history induced by the preparation procedure.     

Viscoelastic evaluation of topical creams containing microcrystalline cellulose/sodium carboxymethyl cellulose as stabilizer

The purpose of this study was to examine the viscoelastic properties of topical creams containing various concentrations of microcrystalline cellulose and sodium carboxymethyl cellulose (Avicel(R) CL-611) as a stabilizer. Avicel CL-611 was used at 4 different levels (1%, 2%, 4%, and 6% dispersion) to prepare topical creams, and hydrocortisone acetate was used as a model drug. The viscoelastic properties such as loss modulus (G"), storage modulus (G'), and loss tangent (tan delta) of these creams were measured using a TA Instruments AR 1000 Rheometer and compared to a commercially available formulation. Continuous flow test to determine the yield stress and thixotropic behavior, and dynamic mechanical tests for determining the linear viscosity time sweep data, were performed. Drug release from the various formulations was studied using an Enhancer TM Cell assembly. Formulations containing 1% and 2% Avicel CL-611 had relative viscosity, yield stress, and thixotropic values that were similar to those of the commercial formulation. The elastic modulus (G') of the 1% and 2% formulation was relatively high and did not cross the loss modulus (G"), indicating that the gels were strong. In the commercial formulation, G' increased after preshearing and broke down after 600 seconds. The strain sweep tests showed that for all formulations containing Avicel CL-611, the G' was above G" with a good distance between them. The gel strength and the predominance of G' can be ranked 6% > 4% > 2%. The strain profiles for the 1% and 2% formulations were similar to those of the commercial formulation. The delta values for the 1% and 2% formulations were similar, and the formulations containing 4% Avicel CL-611 had lower delta values, indicating greater elasticity. Drug release from the commercial preparation was fastest compared to the formulations prepared using Avicel CL-611, a correlation with the viscoelastic properties. It was found that viscoelastic data, especially the strain sweep profiles of products containing Avicel CL-611 1% and 2%, correlated with the commercial formulation. Rheological tests that measure the viscosity, yield stress, thixotropic behavior, other oscillatory parameters such as G' and G" are necessary tools in predicting performance of semisolids.     

Biomechanics of isolated tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides

The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed.