Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.     

Evaluation of Stem Cell-to-Tenocyte Differentiation By Atomic Force Microscopy to Measure Cellular Elastic Moduli

In the present study, we evaluated whether stem cell-to-tenocyte differentiation could be evaluated via measurement of the mechanical properties of the cell. We used mechanical uniaxial cyclic stretching to induce the differentiation of human bone marrow mesenchymal stem cells into tenocytes. The cells were subjected to cyclic elongation of 10 or 15 % at a cyclic frequency of 1 Hz for 24 or 48 h, and differentiation was assessed by real-time PCR (rtPCR) determination of messenger RNA expression levels for four commonly used markers of stem cell-to-tenocyte differentiation: type I collagen, type III collagen, tenascin-C, and scleraxis. The rtPCR results showed that cells subjected to 10 % cyclic elongation for 24 or 48 h differentiated into tenocytes. Atomic force microscopy (AFM) was then used to measure the force curves around the cell nuclei, and the AFM data were used to calculate the elastic moduli of the cell surfaces. The elastic modulus values of the control (non-stretched) cells differed significantly from those of cells stretched at 10 % for 24 or 48 h (P < 0.01). Confocal fluorescence microscopic observations of actin stress fibers suggested that the change in elastic modulus was ascribable to the development of the cellular cytoskeleton during the differentiation process. Therefore, we conclude that the atomic force microscopic measurement of the elastic modulus of the cell surface can be used to evaluate stem cell-to-tenocyte differentiation.     

Mechanical properties of porcine brain tissue in vivo and ex vivo estimated by MR elastography

The mechanical properties of brain tissue in vivo determine the response of the brain to rapid skull acceleration. These properties are thus of great interest to the developers of mathematical models of traumatic brain injury (TBI) or neurosurgical simulations. Animal models provide valuable insight that can improve TBI modeling. In this study we compare estimates of mechanical properties of the Yucatan mini-pig brain in vivo and ex vivo using magnetic resonance elastography (MRE) at multiple frequencies. MRE allows estimations of properties in soft tissue, either in vivo or ex vivo, by imaging harmonic shear wave propagation. Most direct measurements of brain mechanical properties have been performed using samples of brain tissue ex vivo. It has been observed that direct estimates of brain mechanical properties depend on the frequency and amplitude of loading, as well as the time post-mortem and condition of the sample. Using MRE in the same animals at overlapping frequencies, we observe that porcine brain tissue in vivo appears stiffer than porcine brain tissue samples ex vivo at frequencies of 100 Hz and 125 Hz, but measurements show closer agreement at lower frequencies.     

Squeezing and detachment of living cells

The interaction of living cells with their environment is linked to their adhesive and elastic properties. Even if the mechanics of simple lipid membranes is fairly well understood, the analysis of single cell experiments remains challenging in part because of the mechanosensory response of cells to their environment. To study the mechanical properties of living cells we have developed a tool that borrows from micropipette aspiration techniques, atomic force microscopy, and the classical Johnson-Kendall-Roberts test. We show results from a study of the adhesion properties of living cells, as well as the elastic response and relaxation. We present models that are applied throughout the different stages of an experiment, which indicate that the contribution of the different components of the cell are active at various stages of compression, retraction, and detachment. Finally, we present a model that attempts to elucidate the surprising logarithmic relaxation observed when the cell is subjected to a given deformation.     

The effect of composition and microstructure on the viscoelastic properties of dermis

Dermis is a heterogeneous tissue in which extracellular matrix components change in relative amount and spatial assembly across the tissue thickness. The effect of the microstructural and compositional heterogeneities on the overall mechanical response of dermis is, however, largely ignored. In this work, we aimed at gaining a better insight into the effect of extracellular matrix microstructure and composition on the mechanical behaviour of different dermal strata by combining mechanical analysis and selective enzymatic digestion of extracellular matrix components. The dynamical–mechanical tests we performed on bovine dermal splits show that the upper dermal stratum, which is richer in papillary dermis, is characterized by higher mechanical properties than the lower one, which is almost composed of reticular dermis. Moreover, the depletion of interfibrillar proteins, proteoglycans and glycosamminoglycans decreases the dynamic moduli of dermis, especially at small frequencies. Of the two dermal layers tested, the upper dermal layer is more sensitive to the enzymatic treatment than the lower layer. Interestingly, the disruption of the elastic network greatly influenced the viscoelastic properties of upper dermis, inducing a dramatic decrease of both storage (G′) and loss (G″) moduli, suggesting that the spatial assembly of the elastin and collagen networks as well as their mutual interactions dominate the dynamical mechanical response of the tissue.     

Dynamic viscoelasticity of actin cross-linked with wild-type and disease-causing mutant alpha-actinin-4

The actin cross-linker α-actinin-4 has been found to be indispensable for the structural and functional integrity of podocytes; deficiency or alteration of this protein due to mutations results in kidney disease. To gain insight into the effect of the cross-linker on cytoskeletal mechanics, we studied the macroscopic rheological properties of actin networks cross-linked with wild-type and mutant α-actinin-4. The frequency-dependent viscoelasticity of the networks is characterized by an elastic plateau at intermediate frequencies, and relaxation toward fluid properties at low frequencies. The relaxation frequencies of networks with mutant α-actinin-4 are an order of magnitude lower than that with the wild-type, suggesting a slower reaction rate for the dissociation of actin and α-actinin for the mutant, consistent with a smaller observed equilibrium dissociation constant. This difference can be attributed to an additional binding site that is exposed as a result of the mutation, and can be interpreted as a difference in binding energy barriers. This is further supported by the Arrhenius-like temperature dependence of the relaxation frequencies.     

Mechanosensitivity of a Rapid Bioluminescence Reporter System Assessed by Atomic Force Microscopy

Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s⁻¹, the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s⁻¹, indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties.     

Visco- and poroelastic contributions of the zona pellucida to the mechanical response of oocytes

Probing mechanical properties of cells has been identified as a means to infer information on their current state, e.g. with respect to diseases or differentiation. Oocytes have gained particular interest, since mechanical parameters are considered potential indicators of the success of in vitro fertilisation procedures. Established tests provide the structural response of the oocyte resulting from the material properties of the cell’s components and their disposition. Based on dedicated experiments and numerical simulations, we here provide novel insights on the origin of this response. In particular, polarised light microscopy is used to characterise the anisotropy of the zona pellucida, the outermost layer of the oocyte composed of glycoproteins. This information is combined with data on volumetric changes and the force measured in relaxation/cyclic, compression/indentation experiments to calibrate a multi-phasic hyper-viscoelastic model through inverse finite element analysis. These simulations capture the oocyte’s overall force response, the distinct volume changes observed in the zona pellucida, and the structural alterations interpreted as a realignment of the glycoproteins with applied load. The analysis reveals the presence of two distinct timescales, roughly separated by three orders of magnitude, and associated with a rapid outflow of fluid across the external boundaries and a long-term, progressive relaxation of the glycoproteins, respectively. The new results allow breaking the overall response down into the contributions from fluid transport and the mechanical properties of the zona pellucida and ooplasm. In addition to the gain in fundamental knowledge, the outcome of this study may therefore serve an improved interpretation of the data obtained with current methods for mechanical oocyte characterisation.

     

Mechanical properties of plant cell walls probed by relaxation spectra

Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild type. This may be due to the plant's ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, BayesRelax, that deduces relaxation spectra from appropriate rheological measurements is presented and made accessible through a Web interface. BayesRelax models the cell wall as a continuum of relaxing elements, and the ability of the method to resolve small differences in cell wall mechanical properties is demonstrated using tuber tissue from wild-type and transgenic potatoes (Solanum tuberosum) that differ in rhamnogalacturonan I side chain structure.     

Real-time impedance analysis of host cell response to meningococcal infection

Measuring cell proliferation and cell death during bacterial infection involves performing end-point assays that represent the response at a single time point. A new technology from Roche Applied Science and ACEA Biosciences allows continuous monitoring of cells in real-time using specialized cell culture microplates containing micro-electrodes. The xCELLigence system enables continuous measurement and quantification of cell adhesion, proliferation, spreading, cell death and detachment, thus creating a picture of cell function during bacterial infection. Furthermore, lag and log phases can be determined to estimate optimal times to infect cells.In this study we used this system to provide valuable insights into cell function in response to several virulence factors of the meningitis causing pathogen Neisseria meningitidis, including the lipopolysaccharide (LPS), the polysaccharide capsule and the outer membrane protein Opc. We observed that prolonged time of infection with pathogenic Neisseria strains led to morphological changes including cell rounding and loss of cell-cell contact, thus resulting in changed electrical impedance as monitored in real-time. Furthermore, cell function in response to 14 strains of apathogenic Neisseria spp. (N. lactamica and N. mucosa) was analyzed. In contrast, infection with apathogenic N. lactamica isolates did not change electrical impedance monitored for 48 h. Together our data show that this system can be used as a rapid monitoring tool for cellular function in response to bacterial infection and combines high data acquisition rates with ease of handling.     

Wall extensibility and cell hydraulic conductivity decrease in enlarging stem tissues at low water potentials

Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low ψ_w) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low ψ_w by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low ψ_w. It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low ψ_w but that the elastic properties of the walls were of little consequence in this response.     

Effect of Age and Cytoskeletal Elements on the Indentation-Dependent Mechanical Properties of Chondrocytes

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15–2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.     

A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?

Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells. The interpretation of atomic force microscopy-based indentation tests, however, is highly dependent on the use of an appropriate theoretical model of the testing configuration. In this study, a novel, thin-layer viscoelastic model for stress relaxation was developed to quantify the mechanical properties of chondrosarcoma cells in different configurations to examine the hypothesis that viscoelastic properties reflect the metastatic potential and invasiveness of the cell using three well-characterized human chondrosarcoma cell lines (JJ012, FS090, 105KC) that show increasing chondrocytic differentiation and decreasing malignancy, respectively. Single-cell stress relaxation tests were conducted at 2 h and 2 days after plating to determine cell mechanical properties in either spherical or spread morphologies and analyzed using the new theoretical model. At both time points, JJ012 cells had the lowest moduli of the cell lines examined, whereas FS090 typically had the highest. At 2 days, all cells showed an increase in stiffness and a decrease in apparent viscosity compared to the 2-h time point. Fluorescent labeling showed that the F-actin structure in spread cells was significantly different between FS090 cells and JJ012/105KC cells. Taken together with results of previous studies, these findings indicate that cell transformation and tumorigenicity are associated with a decrease in cell modulus and apparent viscosity, suggesting that cell mechanical properties may provide insight into the metastatic potential and invasiveness of a cell.     

Nonlinear Viscoelasticity of Actin Transiently Cross-linked with Mutant α-Actinin-4

Filamentous actin and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. They can also play a critical role in disease; for example, mutations in α-actinin-4 (Actn4), a dynamic actin cross-linking protein, cause proteinuric disease in humans and mice. Amino acid substitutions strongly affect the binding affinity and protein structure of Actn4. To study the physical impact of such substitutions on the underlying cytoskeletal network, we examine the bulk mechanical behavior of in vitro actin networks cross-linked with wild-type and mutant Actn4. These networks exhibit a complex viscoelastic response and are characterized by fluid-like behavior at the longest timescales, a feature that can be quantitatively accounted for through a model governed by dynamic cross-linking. The elastic behavior of the network is highly nonlinear, becoming much stiffer with applied stress. This nonlinear elastic response is also highly sensitive to the mutations of Actn4. In particular, we observe that actin networks cross-linked with Actn4 bearing the disease-causing K255E mutation are more brittle, with a lower breaking stress in comparison to networks cross-linked with wild-type Actn4. Furthermore, a mutation that ablates the first actin binding site (ABS1) in Actn4 abrogates the network's ability to stress-stiffen is standard nomenclature. These changes in the mechanical properties of actin networks cross-linked with mutant Actn4 may represent physical determinants of the underlying disease mechanism in inherited focal segmental glomerulosclerosis.     

Growth Physics in Nitella: a Method for Continuous in Vivo Analysis of Extensibility Based on a Micro-manometer Technique for Turgor Pressure

The view that the plant cell grows by the yielding of the cell wall to turgor pressure can be expressed in the equation: rate = cell extensibility × turgor. All growth rate responses can in principle be resolved into changes in the 2 latter variables. Extensibility will relate primarily to the yielding properties of the cell wall, turgor primarily to solute uptake or production. Use of this simple relationship in vivo requires that at least 2 of the 3 variables be measured in a growing cell. Extensibility is not amenable to direct measurement. Data on rate and turgor for single Nitella cells can, however, be continuously gathered to permit calculation of extensibility (rate/turgor). Rate is accurately obtained from measurements on time-lapse film. Turgor is estimated in the same cell, to within 0.1 atm or less, by measurement of the ability of the cell to compress gas trapped in the closed end of a capillary the open end of which is in the cell vacuole. The method is independent of osmotic equilibrium. It operates continuously for several days, over a several fold increase in cell length, and has response time of less than one minute. Rapid changes in turgor brought on by changes in tonicity of the medium, show that extensibility, as defined above, is not constant but has a value of zero unless the cell has about 80% of normal turgor. Because elastic changes are small, extensibility relates to growth. Over long periods of treatment in a variety of osmotica the threshold value for extensibility and growth is seen to fall to lower values to permit resumption of growth at reduced turgor. A brief period of rapid growth (5× normal) follows the return to normal turgor. All variables then become normal and the cycle can be repeated. The cell remains essentially at osmotic equilibrium, even while growing at 5× the normal rate. The method has potential for detailed in vivo analyses of “wall softening.”     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Effect of Matrix on Cardiomyocyte Viscoelastic Properties in 2D Culture

Cardiomyocyte phenotype changes significantly in 2D culture systems depending on the substrate composition and organization. Given the variety of substrates that are used both for basic cardiac cell culture studies and for regenerative medicine applications, there is a critical need to understand how the different matrices influence cardiac cell mechanics. In the current study, the mechanical properties of neonatal rat cardiomyocytes cultured in a subconfluent layer upon aligned and unaligned collagen and fibronectin matrices were assessed over a two week period using atomic force microscopy. The elastic modulus was estimated by fitting the Hertz model to force curve data and the percent relaxation was determined from stress relaxation curves. The Quasilinear Viscoelastic (QLV) and Standard Linear Solid (SLS) models were fit to the stress relaxation data. Cardiomyocyte cellular mechanical properties were found to be highly dependent on matrix composition and organization as well as time in culture. It was observed that the cells stiffened and relaxed less over the first 3 to 5 days in culture before reaching a plateau in their mechanical properties. After day 5, cells on aligned matrices were stiffer than cells on unaligned matrices and cells on fibronectin matrices were stiffer than cells on collagen matrices. No such significant trends in percent relaxation measurements were observed but the QLV model fit the data very well. These results were correlated with observed changes in cellular structure associated with culture on the different substrates and analyzed for cell-to-cell variability.     

Mechanical properties of the porcine growth plate vary with developmental stage

The objectives of this study were to extract the intrinsic mechanical properties of the growth plate at four different stages of growth and to compare two different methods of extracting these properties. Porcine distal ulnar growth plate samples were obtained from newborn, 4-, 8-, and 18-week (W) pigs and were tested using stress relaxation tests under unconfined compression. A four-parameter curve fitting procedure was developed to extract mechanical properties using the Transversely Isotropic Biphasic Elastic model(TIBPE) (Cohen et?al. in J Biomech Eng Trans Asme 120(4):491–496, 1998) and the Differential Evolution (DE) optimization algorithm (Price et?al. Natural computing series, Springer, Germany 2005). Optimization was done on all experimental curves for the first method and on one average experimental curve per developmental stage in the second. The 4-week stage was studied in two subgroups (a) and (b) due to distinct differences in mechanical properties. Intrinsic mechanical properties of the growth plate varied nonlinearly with developmental stage. Both methods showed that transverse and out-of-plane Young’s moduli (E ₁, E ₃) decrease with developmental stage, whereas transverse permeability (k ₁) increases. The exception is a sharp increase in stiffness and reduction in permeability at the 4W(a) stage, which may be associated with rapid porcine developmental changes at the 3–4 week period. The second method provides a more reliable representation of the average mechanical behavior, whereas the first method allows statistical comparison of optimized mechanical properties. This study characterizes, for the first time, the variation in growth plate mechanical properties for the same animal (porcine) and bone (ulna) model with developmental stage and provides new insight into the progression of musculoskeletal diseases during growth spurts in response to mechanical loading.     

Mechanical difference between white and gray matter in the rat cerebellum measured by scanning force microscopy

The mechanical properties of tissues are increasingly recognized as important cues for cell physiology and pathology. Nevertheless, there is a sparsity of quantitative, high-resolution data on mechanical properties of specific tissues. This is especially true for the central nervous system (CNS), which poses particular difficulties in terms of preparation and measurement. We have prepared thin slices of brain tissue suited for indentation measurements on the micrometer scale in a near-native state. Using a scanning force microscope with a spherical indenter of radius ～20 μm we have mapped the effective elastic modulus of rat cerebellum with a spatial resolution of 100 μm. We found significant differences between white and gray matter, having effective elastic moduli of K=294±74 and 454±53 Pa, respectively, at 3 μm indentation depth (n_g=245, n_w=150 in four animals, p<0.05; errors are SD). In contrast to many other measurements on larger length scales, our results were constant for indentation depths of 2–4 μm indicating a regime of linear effective elastic modulus. These data, assessed with a direct mechanical measurement, provide reliable high-resolution information and serve as a quantitative basis for further neuromechanical investigations on the mechanical properties of developing, adult and damaged CNS tissue.