Local Elastic Modulus Research Articles

Correlation between in vitro expansion-related cell stiffening and differentiation potential of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are an attractive cell source for tissue regeneration, given their self-renewal and multilineage potential. However, they are present in only small percentages in human bone marrow, and are generally propagated in vitro prior to downstream use. Previous work has shown that hMSC propagation can lead to alterations in cell behavior and differentiation potency, yet optimization of differentiation based on starting cell elastic modulus is an area still under investigation. To further advance the knowledge in this field, hMSCs were cultured and routinely passaged on tissue-culture polystyrene to investigate the correlation between cell stiffening and differentiation potency during in vitro aging. Local cell elastic modulus was measured at every passage using atomic force microscopy indentation. At each passage, cells were induced to differentiate down myogenic and osteogenic paths. Cells induced to differentiate, as well as undifferentiated cells were assessed for gene and protein expression using quantitative polymerase chain reaction and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Myogenic and osteogenic cell potential are highly reliant on the elastic modulus of the starting cell population (of undifferentiated cells), and this potential appears to peak when the innate cell elastic modulus is close to that of differentiated tissue. However, the latent expression of the same markers in undifferentiated cells also appears to undergo a correlative relationship with cell elastic modulus, indicating some endogenous effects of cell elastic modulus and gene/protein expression. Overall, this study correlates age-related changes with regards to innate cell stiffening and gene/protein expression in commercial hMSCs, providing some guidance as to maintenance and future use of hMSCs in future tissue engineering applications.

Temperature dependent stacking fault energy of FeCrCoNiMn high entropy alloy

The stacking fault energy (SFE) of paramagnetic FeCrCoNiMn high entropy alloy is investigated as a function of temperature via ab initio calculations. We divide the SFE into three major contributions: chemical, magnetic and strain parts. Structural energies, local magnetic moments and elastic moduli are used to estimate the effect of temperature on each term. The present results explain the recently reported twinning observed below room-temperature and predict the occurrence of the hexagonal phase at cryogenic conditions.

Spatial Heterogeneity of Glassy Polymer Films

By studying the morphology of polystyrene films subjected to a fast structuration method, we demonstrate the spatial heterogeneity of their surface viscoelasticity at temperatures well below the glass transition temperature, Tg. Our results point to a nonrandom arrangement of zones of different elastic modulus deep in the glassy state. The spatial distribution of viscoelastic properties of the film was temperature dependent; we observed the presence of soft zones (surrounded by areas of larger elastic modulus) spanning increasingly larger areas of the surface as T gets closer to Tg. The heterogeneity on viscoelastic properties of the film is supported by the temporal evolution and relaxation of the structured surfaces and by direct measurements of the spatial distribution of the local elastic modulus of polystyrene films.

Characterization of local elastic modulus in confined polymer films via AFM indentation.

The properties of polymers near an interface are altered relative to their bulk value due both to chemical interaction and geometric confinement effects. For the past two decades, the dynamics of polymers in confined geometries (thin polymer film or nanocomposites with high-surface area particles) has been studied in detail, allowing progress to be made toward understanding the origin of the dynamic effects near interfaces. Observations of mechanical property enhancements in polymer nanocomposites have been attributed to similar origins. However, the existing measurement methods of these local mechanical properties have resulted in a variety of conflicting results on the change of mechanical properties of confined polymers. Here, an atomic force microscopy (AFM)-based method is demonstrated that directly measures the mechanical properties of polymers adjacent to a substrate with nanometer resolution. This method allows us to consistently observe the gradient in mechanical properties away from a substrate in various materials systems, and paves the way for a unified understanding of thermodynamic and mechanical response of polymers. This gradient is both longer (up to 170 nm) and of higher magnitude (50% increase) than expected from prior results. Through the use of this technique, we will be better able to understand how to design polymer nanocomposites and polymeric structures at the smallest length scale, which affects the fields of structures, electronics, and healthcare.

Judging diatoms by their cover: variability in local elasticity of Lithodesmium undulatum undergoing cell division.

Unique features of diatoms are their intricate cell covers (frustules) made out of hydrated, amorphous silica. The frustule defines and maintains cell shape and protects cells against grazers and pathogens, yet it must allow for cell expansion during growth and division. Other siliceous structures have also evolved in some chain-forming species as means for holding neighboring cells together. Characterization and quantification of mechanical properties of these structures are crucial for the understanding of the relationship between form and function in diatoms, but thus far only a handful of studies have addressed this issue. We conducted micro-indentation experiments, using atomic force microscopy (AFM), to examine local variations in elastic (Young's) moduli of cells and linking structures in the marine, chain-forming diatom Lithodesmium undulatum. Using a fluorescent tracer that is incorporated into new cell wall components we tested the hypothesis that new siliceous structures differ in elastic modulus from their older counterparts. Results show that the local elastic modulus is a highly dynamic property. Elastic modulus of stained regions was significantly lower than that of unstained regions, suggesting that newly formed cell wall components are generally softer than the ones inherited from the parent cells. This study provides the first evidence of differentiation in local elastic properties in the course of the cell cycle. Hardening of newly formed regions may involve incorporation of additional, possibly organic, material but further studies are needed to elucidate the processes that regulate mechanical properties of the frustule during the cell cycle.

Structural Size Effect on Mechanical Behavior of Intermetallic Material in Solder Joints: Experimental Investigation

The elastic and plastic mechanical properties of intermetallic compound (IMC) phases of a lead-free Sn3.5Ag/Cu-substrate soldering system are investigated in different sized joints using nano-indentation. The specimens were prepared using solid–liquid interdiffusion soldering process with joint sizes ranging from 15 to 450 μm. Solder joints were subjected to 360 °C soldering temperature for 20 min and then air cooled to room temperature to create testable IMCs thicknesses. Nano-indentation was used to extract the elastic and plastic properties of Cu6Sn5, Cu3Sn, and Ag3Sn IMCs and β-tin and copper materials. Cu–Sn IMCs formed in specimens with smaller joint size show higher elastic modulus, hardness, and yield strength and lower work hardening exponent. This was attributed to the dimensional constraints associated with decreasing joint size and the local stresses developed during fabrication in joints with different sizes. Local elastic modulus and hardness of single grains of Cu6Sn5 were obtained using a combination of nano-indentation and electron backscatter diffraction (EBSD) techniques. Grains with c-axis at a 45 deg angle with respect to the nano-indentation loading direction show higher elastic modulus (∼8.70% higher) and hardness (8.85% higher) compared to the grains that have a c-axis that is almost perpendicular to the nano-indentation loading direction.

Abstract 230: Regional Variation in Intrinsic Mechanics of Arterial Vascular Smooth Muscle Cells in Spontaneously Hypertensive Rats

Aims: An increased aortic stiffness is a fundamental manifestation of hypertension (HT). Our previous study showed that intrinsic mechanical properties of aortic vascular smooth muscle cells (VSMC) are an important contributor to the increased large artery stiffness in HT. However, whether VSMC mechanics of smaller arteries is also altered in HT remains unknown. The goal of this study is to test our hypothesis that the VSMC elastic and adhesive properties vary along the arterial tree reflecting the regional heterogeneities of physiological loading and geometric properties of the artery wall. Methods and Results: Primary VSMCs were isolated from the thoracic aorta (TA) and renal artery (RA) of adult spontaneously hypertensive rats (SHR) (16 weeks old, male) and age-matched Wistar-Kyoto normotensive (WKY) rats. Atomic force microscopy (AFM) was used to measure mechanical properties of individual VSMC at 37°C. Local apparent elastic modulus (Eap) was determined using Hertz contact analysis for a cone to model the indentation force curve, and maximum adhesion force (Fad) was obtained from the retraction force curve; results were shown as mean (±SD) (n=10 cells per condition) and compared using two way ANOVA. Eap of VSMCs from the TA was significantly higher in SHR (7.0± 1.3 kPa) vs. WKY (5.3 ± 1.5 kPa; p < 0.001) and Fad was significantly larger in SHR (39.9 7.2 pN) vs. WKY (30.6 10.9 pN; p < 0.01). No difference was found between SHR and WKY in VSMCs from renal artery in terms of Eap (4.5 0.8 kPa vs. 4.8 0.9 kPa; p = 0.59) and Fad (23.1 5.2 pN vs. 28.0 7.0 pN; p = 0.17). Although no difference was observed in cell shape represented by the cellular length:width ratio (p > 0.25), stiffness and adhesion of VSMC from TA were significantly higher vs. RA in SHR (p<0.0001) but not in WKY (P>0.35), indicating that the altered mechanics of VSMC in hypertension is more prominent in the large conduction vessel compared to the small artery. Conclusion: Intrinsic stiffness and adhesion of isolated VSMC are elevated preferentially in the thoracic aorta of SHR rats; the regional variations may associate mechanistically with increased aortic tissue stiffness in hypertension.

Stabilizing the Microphase Separation of Block Copolymers by Controlled Photo‐crosslinking

Photo‐crosslinkable material is produced via self‐assembly of poly(methyl methacrylate)‐block‐poly(n‐butyl acrylate) (PMMA‐b‐PBA) block copolymers, in which the MMA block is decorated with coumarin moieties. The block copolymer is synthesized by reversible addition‐fragmentation chain transfer (RAFT) polymerization. Atomic force microscopy (AFM) reveals microphase separation into nanodomains of controlled and regular morphology. The local elastic modulus of different nanodomains is determined using the PeakForce Quantitative Nano‐Mechanics (PFQNM) mode of AFM. The global modulus, as determined by mechanical spectroscopy, agrees well with the local values. The crosslinking of the block copolymer, via photodimerization of the incorporated coumarin moieties using UV light, is traced by UV–vis absorption measurements and resulted in an increase of the global elastic modulus. The crosslinking reaction proceeding in the solid state is surprisingly effective and stabilizes the morphology to such an extent that equilibrium phase separation, as targeted by annealing, is effectively suppressed. image

Endodontic instruments after torsional failure: Nanoindentation test

This study aimed to evaluate effects of torsional loading on the mechanical properties of endodontic instruments using the nanoindentation technique. ProFile (PF; size 30, taper 04; Dentsply Maillefer, Switzerland) and stainless steel (SS; size 30, taper 02; Mani, Japan) instruments were subjected to torsional test. Nanoindentation was then performed adjacent to the edge of fracture (edge) and at the cutting part beside the shank (shank). Hardness and elastic modulus were measured under 100-mN force on 100 locations at each region, and compared to those obtained from the same regions on new instruments. It showed that PF and SS instruments failed at 559 ± 67 and 596 ± 73 rotation degrees and mean maximum torque of 0.90 ± 0.07 and 0.99 ± 0.05 N-cm, respectively. Hardness and elastic modulus ranged 4.8-6.7 and 118-339 GPa in SS, and 2.7-3.2 and 52-81 GPa in PF. Significant differences between torsion-fractured and new instruments in hardness and elastic modulus were detected in the SS system used. While in PF system, the edge region after torsional fracture had significantly lower hardness and elastic modulus compared to new instruments. The local hardness and modulus of elasticity of endodontic instruments adjacent to the fracture edge are significantly reduced by torsional loading.

Local elasticity and mobility of twin boundaries in martensitic films studied by atomic force acoustic microscopy

Nanoscale elastic properties of twinned martensite NiMnGa films were characterized by means of atomic force acoustic microscopy using cantilever contact-resonance spectra to measure the local contact stiffness k* and the local damping Q−1, which contains information on the crystallographic anisotropy of martensitic twin variants and the dissipative motion of twin boundaries (TBs). Images of k* and indentation modulus maps were obtained. Similar to topography images measured by conventional atomic force microscopy in contact mode, they show the nature of the twin structure and thus a regular variation in local elastic modulus. A correlation between k* and Q−1 was observed and mirrors the motion of the TB accompanied by a viscoelastic procedure. The k*-image and the topography image measured are opposite in contrast, which likely arises from mobile and immobile TBs depending on the geometry of twinning. Multi-resonance spectra were measured, which can be related to martensitic multivariants and are explainable as different types of nanotwins. A critical stress, defined as the starting point of softening due to TB movement was determined to be about 0.5 GPa for a thick film (1 μm) and 0.75 GPa for a thin film (0.15 μm), respectively. The values are much larger than that measured for bulk materials, but reasonable due to a large internal stress in the films.

Nanoscale elasticity mappings of micro-constituents of abalone shell by band excitation-contact resonance force microscopy

The macroscopic mechanical properties of the abalone shell have been studied extensively in the literature, but the in situ nanoscale elasticity of various micro-constituents in the shell have not been characterized and reported yet. In this study, the nanoscale elasticity mappings including different micro-constituents in abalone shell were observed by using the Contact Resonance Force Microscopy (CR-FM) technique. CR-FM is one of the advanced scanning probe microscopy techniques that is able to quantify the local elastic moduli of various materials in a non-destructive manner. Instead of an average value, an elasticity mapping that reveals the nanoscale variations of elastic moduli with location can be extracted and correlated with the topography of the structure. Therefore in this study, by adopting the CR-FM technique that is incorporated with the band excitation technique, the elasticity variations of the abalone shell caused by different micro-constituents and crystal orientations are reported, and the elasticity values of the aragonite and calcite nanograins are quantified.

Interpretation of Results of Penetration Tests Performed on Timber Structures in Bending

The paper concentrates on the determination of local elastic moduli of timber in the fiber direction. To that end a single commercially produced glued timber beam was subjected to 3600 penetration measurements. The beam was first covered by a regular grid of monitoring points at which the depth of indentation was measured. The pin was shot into the wood with a given energy (Pilodin 6J). At the same time the moisture content at 80 selected points was determined. A specific measurement order was assumed to exclude principal measurement errors on the one hand and to minimize systematic measurement errors on the other. We expect the measured elastic moduli to serve as an input for advanced finite element simulations on the bases of stochastic analysis. In such a case the local measured moduli represent in a given segment of each lamella an ensemble of data characterized by a selected probability distribution. These distributions are then employed in the LHS based stochastic simulation to provide probability distribution of the maximum deflection for a given load level. Apart from that it appears meaningful to compare independently the probability distributions of the elastic moduli for segments of the lamella (these may considerably differ owing to the specifics of the production of structures made from glued lamella timber) with statistical data from the whole beam. Based on the measured data the correlation matrix relating statistical dependence of individual segments can be estimated thus improving the quality of the stochastic model.

Double side friction stir welding of AA6082 sheets: Microstructure and nanoindentation characterization

Friction stir welding (FSW) of aluminum alloys is currently used in modern automotive and transportation industry. The welded sheets must possess adequate elastic–plastic response and formability levels similar to that of the base alloy sheet. In the last few years different FSW processing and configurations have been proposed. In this study, a double side friction stir welding (DS-FSW) process was compared to conventional pin and pinless FSW of AA6082 sheet. The microstructure modifications and the local mechanical response, namely, hardness and elastic modulus, were here investigated. Nanoindentation was used to mechanically characterize the different welded zones of interest, the thermomechanical heat affected zone (TMAZ), the stirred zone (SZ), in the advancing and the retreating side, at different sheet section depths. The better microstructure uniformity, at the stirred zone, and the closer hardness and elastic modulus values to those of the base metal can explain the better formability showed by the DS-FSW, with respect to the conventional FSW.

Finite Element Modeling to Simulate the Elasto-Plastic Behavior of Polycrystalline in 718

A 3D strain gradient plasticity finite element model was developed to simulate the elasto-plastic behavior of polycrystalline IN 718 alloys. The proposed model constructed in the basis of the so-called Kocks-Mecking model is used to determine the influence of microstructure attributes on the inelastic stress-strain distribution. Representative Volume Elements (RVEs) of different edge size but similar grain morphology and affordable computational meshes were tested to investigate the link between micro and macro variables of deformation and stress. The virtual specimens subjected to continuous monotonic straining loading conditions were constrained with random periodic boundary conditions. The difference in crystallographic orientation (which evolves in the process of straining) and the incompatibility of deformation between neighboring grains were accounted by the introduction of averaged Taylor factors and the evolution of geometrically necessary dislocation density. The effect of plastic deformation gradients imposed by the microstructure is clearly observed. Results demonstrate a strong dependence of flow stress and plastic strain on phase type and grain size. A main strategy for constitutive modeling of individual bulk grains is presented. The influence of the grain size on the aggregate response, in terms of local stress variations and aggregate elastic moduli was analyzed. It was observed that the elastic modulus in the bulk material is not dependent on grain size.

COMBINED ATOMIC FORCE AND FLUORESCENCE MICROSCOPIES TO MEASURE SUBCELLULAR MECHANICAL PROPERTIES OF LIVE CELLS

Atomic force microscopy (AFM) has been widely applied to study cellular functions;however, the relationship between cellular elasticity and ultrastructure density of a live cell remains to be discovered. The objective of this study was thus to extend our previous method of integrating AFM and immunofluorescence imaging to measure the ultrastructure distribution-related local mechanical properties of live cells. First, the morphology of a live cell was obtained by AFM. Second, the indentation sites were selected and flexible force volume indentation was performed. Third, the immunofluorescence image of the cell was obtained. The last was the mapping of the indentation site to the immunofluorescence image and obtaining the relationship between the local elastic properties and cytoskeleton density. The results on differentiated rat Schwann cells (RSCs) showed that the elastic modulus of stress fibers is higher than those of the nucleus and cytosol. The local elastic modulus of the live RSCs is correlated to the actin density, and the stress fiber that behaves like a pretension beam can give RSCs enough strength to envelop axons during myelination. In particular, the elastic properties of the live RSCs were twofold lower than those of the fixed. The results demonstrated the integrated method's applicability for a live cell.

Analysis of heat-treated bovine cortical bone by thermal gravimetric and nanoindentation

Xenograft bone has been widely used as a bone grafting material because it gains advantages in biological and mechanical properties as compare with the use of an allograft bone. Heat-treatment of bone is recognized as one of the simple and practical methods to lower the human immunodeficiency virus (HIV) infection and overcome the risks of rejection and disease transfer during the bone transplantation. Therefore, understanding the change of bone’s organic matrix after heat treatment has become a significant topic. In this study, thermal gravimetric analysis (TGA) was used to investigate the condition of organic constituents of a bovine cortical bone. In order to well characterize the microstructural and mechanical property of the bone after heat treatment, nanoindention technique was also employed to measure the localized elastic modulus (E) and hardness (H) of its interstitial lamellae and osteons lamellae at the temperatures of 23°C (RT), 37°C, 90°C, 120°C and 160°C, respectively.The TGA results demonstrated that heat-treated bones had three stages of weight loss. The first stage was the loss of water, which started from RT to 160°C. Follow by a weight loss of organic constituents starting from 200°C to 600°C. Upon reaching 600°C, the organic constituents were decomposed and mineral phase loss started taking place until 850°C. From the nanoindentation results, it showed the values of E and H measured for the interstitial lamellae were higher than that of the osteons lamellae. This phenomenon indicates that the interstitial lamellae are stiffer and easy to be mineralized than osteons lamellae. For a specimen heat-treated at 90°C, the values of E and H of interstitial lamellae and osteons lamellae were similar to a non-heat-treated specimen. For a specimen heat-treated at 120°C, its interstitial lamellae had higher E and H values than osteons lamellae. When a specimen was heat-treated at 160°C, both interstitial lamellae and osteons lamellae demonstrated a slight decrease of their E and H values. An ANOVA statistical analysis was used to analyze the difference in elastic properties and hardness in various temperature ranges.

Determination of local and global elastic moduli of valve interstitial cells cultured on soft substrates

The elasticity of the extracellular matrix profoundly affects biological responses of cells, but also their mechanical properties. Single cell mechanical properties are often measured by atomic force microscopy (AFM), but technical guidelines for AFM measurement of cells grown on soft substrates are not well established. In this study, the local and global elastic moduli of aortic valve interstitial cells (VICs) cultured on soft polyacrylamide substrates (3–144kPa) were characterized via AFM force mapping using pyramidal and spherical tips, respectively. Local and global VIC modulus values both increased with substrate stiffness (p<0.05), with the average local cell modulus being consistently two to three times greater than the global modulus (p<0.05). For local measurements, a minimum of four measurements was required to observe the trend of increasing cell modulus with substrate stiffness, but there was no advantage to testing additional spots. The Hertz model was relatively accurate in estimating the global cell elastic modulus (<12% error, based on validated finite element analyses), despite the cells being of finite thickness and grown on deformable substrates, neither of which are accounted for in the Hertz model. The results of this study provide practical guidelines for efficient AFM-based measurement of the mechanical properties of cells on gels. They also provide new physiologically-relevant data on VIC mechanical properties and their correlation with substrate stiffness-dependent cytoskeletal changes, with relevance to heart valve mechanobiology and disease.

Measuring spatial distribution of the local elastic modulus in glasses

Glasses exhibit spatially inhomogeneous elastic properties, which can be investigated by measuring their elastic moduli at a local scale. Various methods to evaluate the local elastic modulus have been proposed in the literature. A first possibility is to measure the local stress-local strain curve and to obtain the local elastic modulus from the slope of the curve or, equivalently, to use a local fluctuation formula. Another possible route is to assume an affine strain and to use the applied global strain instead of the local strain for the calculation of the local modulus. Most recently, a third technique has been introduced, which is easy to be implemented and has the advantage of low computational cost. In this contribution, we compare these three approaches by using the same model glass and reveal the differences among them caused by the nonaffine deformations.

Relationship between local and global modulus of elasticity in bending and its consequence on structural timber grading

The study analyses the relationship between local and global modulus of elasticity and develops and evaluates different models to predict local from global modulus measurements. The mechanical tests were performed on four species commonly used in Italy for structural purposes: fir, Douglas-fir, Corsican pine and chestnut. Two or three cross-sections and two provenances were sampled for each species. A theoretical analysis showed that the local–global modulus relationship was of polynomial form with only one coefficient. The effect of the species on the relationship was significant as well as the cross-section but only for softwoods. The effect of the cross-section was explained by the presence and the size of defects in the mid span. The different models were applied and then compared by means of the optimum grading: only slight differences among models emerged. Although optimum grading was strongly dependent on the sampling and on the grade combination, for softwoods the model for species and section showed very similar results to the grading with the true local modulus; inclusion of the knot values in the model led to only slight improvements. For chestnut all models were found to be comparable.

Multi-scale materials data for 3D TSV stack performance simulation and model validation

Within a DFM type of methodology, the application of physics-based models for a multi-scale simulation of mechanical stress in 3D TSV-stacked products was proposed in [1]. Highly accurate multi-scale materials data are needed as input for this approach of simulation and for model validation. This paper focuses on the set of materials data for wafer-level and package-level structures, needed to feed a materials database that comprises the input parameters for simulation. Particularly the generation of materials data such as (local and effective) Young's modulus, Poisson ratio and (effective) coefficients of thermal expansion (CTE) on several scales will be described. The nanoindentation method was developed further to measure local and effective elastic modulus values and Poisson ratio for micro-bump, TSV and BEoL stacks. TCAD simulations show significant influence of the copper microstructure on the charge carrier mobility in transistors [2]. To consider the orientation dependence of elastic properties of copper, nanoindentation with extremely low penetration depths was applied. A high-resolution in-situ technique was developed to determine “composite CTE” values for BEoL stacks. A free-standing cantilever containing the region of interest was extracted using the FIB technique, and imaged in a SEM at several temperatures. For model validation and calibration, local stress measurements are needed to determine the effect of the TSV/package-induced stress on the transistor performance. Due to the high resolution needed, the only direct technique to measure strain in transistor channels is TEM. For the first time, the strain in transistor channels will be reported as a function of the distance from the TSV, based on Convergent Beam Electron Diffraction (CBED) data.