Mechanical behavior of an individual adherent MLO-Y4 osteocyte under shear flow

Mechanical properties of a single cell and its mechanical response under stimulation play an important role in regulating interactions between cell and extracellular matrix and affecting mechanotransduction. Osteocytes exhibit solid-like viscoelastic behavior in response to the interstitial fluid shear resulting from tissue matrix deformation. This study intends to quantitatively describe the mechanical behavior of osteocytes combining in vitro experiment and fluid–structure interaction (FSI) finite element (FE) model. The cell is configured in the FSI FE model using the observed data from quasi-3D images. Instead of simply assigning the cellular viscoelastic parameters by statistical data, the mechanical parameters are determined by an iterative algorithm comparing the experimental and the computational results from the FE model. The viscoelastic parameters of osteocytes are obtained as: the equilibrium elasticity modulus \(k_{1}=0.15\pm 0.038\,\hbox {kPa}\), instantaneous elasticity modulus \((k_{1}+k_{2})=0.77\pm 0.23\,\hbox {kPa}\), viscosity coefficient \(\eta =1.38\pm 0.33\,\hbox {kPa}\,\hbox {s}\). A novel index to quantify the cell adhesion is also put forward. In addition, an interesting competition phenomenon is revealed on the cell surface concerning stress and strain, i.e., the place with high stress has low strain and that with low stress has high strain. The proposed method provides a novel technique to study the mechanical behavior of individual adherent cell in vitro. It is believed that this quantitative technique not only determines cell mechanical behavior but also helps elucidate the mechanism of mechanotransduction in various types of cells.     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Changes in the stiffness of human mesenchymal stem cells with the progress of cell death as measured by atomic force microscopy

This note reports observations of the change of stiffness of human mesenchymal stem cells (hMSCs) with the progress of cell death as measured by AFM. hMSC with impaired membrane, dead and viable cells were labelled with Annexin V and Propidium Iodide after 24 h cold storage, followed by AFM measurement and Young's modulus of cells was derived. Viable hMSCs have a Young's modulus (E) in the range of 0.81–1.13 kPa and consistent measurement was observed when different measurement locations were chosen. E of cells with partially impaired membrane was 0.69±0.17 kPa or in the range of 2.04–4.74 kPa, depending upon the measurement locations. With the loss of membrane integrity, though there was no variation on measured E between different locations, a mixed picture of cell stiffness was observed as indicated by cells with E as low as 0.09±0.03 kPa, in a mid-range of 4.62±0.67 kPa, and the highest of up to 48.98±19.80 kPa. With the progress of cell death, the highest stiffness was noticed for cells showing a more granular appearance; also the lowest stiffness for cells with vacuole appearance. Findings from this study indicate that cell stiffness is significantly altered with the progress of cell death.     

Alteration Young’s moduli by protein 4.1 phosphorylation play a potential role in the deformability development of vertebrate erythrocytes

The mechanical properties of vertebrate erythrocytes depend on their cytoskeletal protein networks. Membrane skeleton proteins spectrin and protein 4.1 (4.1R) cross-link with actin to maintain membrane stability under mechanical stress. Phosphorylation of 4.1R alters the affinity of 4.1R for spectrin–actin binding and this modulates the mechanical properties of human erythrocytes. In this study, phorbol 12-myristate-13-acetate (PMA)-induced phosphorylation of 4.1R was tested, erythrocyte deformability was determined and the erythrocyte elastic modulus was detected in human, chick, frog and fish. Furthermore, amino acid sequences of the functionally important domains of 4.1R were analyzed. Results showed that PMA-induced phosphorylation of 4.1R decreased erythrocyte deformability and this property was stable after 1 h. The values of Young’s modulus alteration gradually decreased from human to fish (0.388±0.035 kPa, 0.219±0.022 kPa, 0.191±0.036 kPa and 0.141±0.007 kPa). Ser-312 and Ser-331 are located within the consensus sequence recognized by protein kinase C (PKC); however, Ser-331 in zebrafish was replaced by Ala-331. The sequence of the 8 aa motif from vertebrate 4.1R showed only one amino acid mutation in frog and numerous substitutions in fish. Analyses of Young’s modulus suggested that the interaction between 4.1R with the spectrin–actin binding domain may have a special relationship with the development of erythrocyte deformability. In addition, amino acid mutations in 4.1R further supported this relationship. Thus, we hypothesize that alteration of membrane skeleton protein binding affinity may play a potential role in the development of erythrocyte deformability, and alteration of Young’s modulus values may provide a method for determining the deformability development of vertebrate erythrocytes.     

Applied osmotic loading for promoting development of engineered cartilage

This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300 mOsM) or isotonic (330 mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400 mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89 kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187 kPa and 6.77±0.54%ww (GAG) for the 400 mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle–van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.     

Creep indentation of single cells

An apparatus for creep indentation of individual adherent cells was designed, developed, and experimentally validated. The creep cytoindentation apparatus (CCA) can perform stress-controlled experiments and measure the corresponding deformation of single anchorage-dependent cells. The apparatus can resolve forces on the order of 1 nN and cellular deformations on the order of 0.1 micron. Experiments were conducted on bovine articular chondrocytes using loads on the order of 10 nN. The experimentally observed viscoelastic behavior of these cells was modeled using the punch problem and standard linear solid. The punch problem yielded a Young's modulus of 1.11 +/- 0.48 kPa. The standard linear solid model yielded an instantaneous elastic modulus of 8.00 +/- 4.41 kPa, a relaxed modulus of 1.09 +/- 0.54 kPa, an apparent viscosity of 1.50 +/- 0.92 kPa-s, and a time constant of 1.32 +/- 0.65 s. To our knowledge, this is the first time that stress-controlled indentation testing has been applied at the single cell level. This methodology represents a new tool in understanding the mechanical nature of anchorage-dependent cells and mechanotransductional pathways.     

Frequency-dependent shear impedance of the tectorial membrane

Microscale mechanical probes were designed and bulk-fabricated for applying shearing forces to biological tissues. These probes were used to measure shear impedance of the tectorial membrane (TM) in two dimensions. Forces were applied in the radial and longitudinal directions at frequencies ranging from 0.01-9 kHz and amplitudes from 0.02-4 μN. The force applied was determined by measuring the deflection of the probes’ cantilever arms. TM impedance in the radial direction had a magnitude of 63 ± 28 mN · s/m at 10 Hz and fell with frequency by 16 ± 0.4 dB/decade, with a constant phase of −72 ± 6°. In the longitudinal direction, impedance was 36 ± 9 mN · s/m at 10 Hz and fell by 19 ± 0.4 dB/decade, with a constant phase of −78 ± 4°. Impedance was nearly constant as a function of force except at the highest forces, for which it fell slightly. These results show that the viscoelastic properties of the TM extend over a significant range of audio frequencies, consistent with a poroelastic interpretation of TM mechanics. The shear modulus G′ determined from these measurements was 17-50 kPa, which is larger than in species with a lower auditory frequency range. This value suggests that hair bundles cannot globally shear the TM, but most likely cause bulk TM motion.     

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.     

Molecular modeling of the axial and circumferential elastic moduli of tubulin

Microtubules play a number of important mechanical roles in almost all cell types in nearly all major phylogenetic trees. We have used a molecular mechanics approach to perform tensile tests on individual tubulin monomers and determined values for the axial and circumferential moduli for all currently known complete sequences. The axial elastic moduli, in vacuo, were found to be 1.25 GPa and 1.34 GPa for α- and β-bovine tubulin monomers. In the circumferential direction, these moduli were 378 MPa for α- and 460 MPa for β-structures. Using bovine tubulin as a template, 269 homologous tubulin structures were also subjected to simulated tensile loads yielding an average axial elastic modulus of 1.10 ± 0.14 GPa for α-tubulin structures and 1.39 ± 0.68 GPa for β-tubulin. Circumferentially the α- and β-moduli were 936 ± 216 MPa and 658 ± 134 MPa, respectively. Our primary finding is that that the axial elastic modulus of tubulin diminishes as the length of the monomer increases. However, in the circumferential direction, no correlation exists. These predicted anisotropies and scale dependencies may assist in interpreting the macroscale behavior of microtubules during mitosis or cell growth. Additionally, an intergenomic approach to investigating the mechanical properties of proteins may provide a way to elucidate the evolutionary mechanical constraints imposed by nature upon individual subcellular components.     

Elastic,permeability and swelling properties of human intervertebral disc tissues: A benchmark for tissue engineering

The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390 kPa) compared to the NP (100 kPa) or AF (30 kPa). The permeability was very different across tissue regions, with the AF permeability (64 E⁻¹⁶ m⁴/N s) higher than the NP and CEP (~5.5 E⁻¹⁶ m⁴/N s). Additionally, a normalized time-constant (3000 s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical behavior and transport.     

Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells

Cells are subjected to static tension of different magnitudes when cultured on substrates with different stiffnesses. It has long been recognized that mechanical stress is an important modulator of the intervertebral disc degeneration. Here we studied the influence of substrate stiffness on cell morphology, apoptosis and extracellular matrix (ECM) metabolism of the rat annulus fibrosus (AF) cells which are known to be mechanosensitive cells. Polyacrylamide gel substrates with three different stiffnesses were prepared by varying the concentration of acrylamide and bisacrylamide, and the elastic modulus of the different gel substrates were measured with atomic force microscopy (AFM). First-passage rat annular cells were cultured on soft, intermediate, rigid substrates or plastics for 24 or 48 h. The percentages of apoptotic cells were detected by flow cytometry and caspase-3 activity, and morphologic changes were visualized by Hoechst 33258 staining and F-actin staining. In addition, the expression of ECM genes (Col1α1, Col2α1, aggrecan, MMP-3, MMP-13 and ADAMTS-5) were analyzed by RT-PCR. The three different substrates had elastic moduli varying between 1 ± 0.23 kPa (soft, 5% gel with 0.06% bis), 32 ± 2.89 kPa (intermediate, 10% gel with 0.13% bis) and 63 ± 3.45 kPa (rigid, 10% gel with 0.26% bis) with a thickness about 60-70 μm. Most of the rat AF cells appeared small and rounded, and lost most of their stress fibers when cultured on soft substrate. There was a significant increase in the percentage of apoptotic cells in the rat AF cells cultured on soft and intermediate substrates relative to those on plastic surface, with a parallel decrease in the area of cell spreading and nucleus. The AF cells grown on intermediate or rigid substrate had reduced expression of Col1α1, Col2α1 and aggrecan and enhanced expression of MMP-3, MMP-13, and ADAMTS-5 at 24 h or 48 h, respectively, relative to those cultured on plastic surface. Conversely, we observed an up-regulation of Col2α1 and aggrecan and no change in the gene expression of MMP-3, MMP-13, and ADAMTS-5 in AF cells on soft substrates. Rat AF cells are sensitive to substrate stiffness which can regulate the morphology, growth, apoptosis and ECM metabolism of rat AF cells, thus indicating the importance of substrate choice for cell transplantation and regeneration for the treatment of disc degeneration using tissue-engineering technique.     

Mechanical properties of native and cross-linked type I collagen fibrils

Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 ± 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 ± 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 ± 7 MPa at ambient conditions and to 3.4 ± 0.2 MPa in phosphate-buffered saline.     

基于原子力显微镜测量内耳螺旋器的弹性特征

应用原子力显微镜分析内耳螺旋器(Corti器)不同部位的弹性特征。采用豚鼠内耳基底膜底回新鲜标本,用原子力显微镜在液相接触式测量,获得不同部位力曲线。经计算,对应Corti器相当于Hensen细胞、外毛细胞、柱细胞、内毛细胞、内指细胞、盖膜的部位及基底膜底面局部,其杨氏模量均值分别为46±1.7、59±0.9、250±31、140±2.8、430±29.9、210±7.2和230±8.8 kPa。结果表明,基底膜径向排列的组织结构不同,杨氏模量存在明显差异,在整块基底膜标本上测量Corti器各结构的杨氏模量能更准确地反映它们在生理状态下的弹性特征。     

Fast fluorescence laser tracking microrheometry. I: instrument development

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(ω). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.     

Early activation of the β-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-3 kinase/Akt, and focal adhesion kinase

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/β-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced β-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7 ± 0.3 Pa, 5 Hz) for 30 min induced β-catenin stabilization and activation of the Wnt/β-catenin signaling pathway. The PFF-induced stabilization of β-catenin and activation of the β-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 μM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 μM). Addition of nitric oxide synthase inhibitor l-NAME (1.0 mM) also abolished PFF-induced stabilization of β-catenin. This suggests that mechanical loading activates the β-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of β-catenin in mechanical adaptation of bone.     

Quantitative investigation of calcimimetic R568 on beta cell adhesion and mechanics using AFM single-cell force spectroscopy

In this study we use a novel approach to quantitatively investigate mechanical and interfacial properties of clonal β-cells using AFM-Single Cell Force Spectroscopy (SCFS). MIN6 cells were incubated for 48 h with 0.5 mM Ca²⁺ ± the calcimimetic R568 (1 μM). AFM-SCFS adhesion and indentation experiments were performed by using modified tipless cantilevers. Hertz contact model was applied to analyse force–displacement (F–d) curves for determining elastic or Young’s modulus (E). Our results show CaSR-evoked increases in cell-to-cell adhesion parameters and E modulus of single cells, demonstrating that cytomechanics have profound effects on cell adhesion characterization.     

Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/−80 Pa), dendritic cells (DCs, 440 +110/−90 Pa), and macrophages (MPHs, 900 +110/−100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young’s modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation.     

Microplate live cell assay system for early events in mechanotransduction

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.     

Flow-induced hardening of endothelial nucleus as an intracellular stress-bearing organelle

The mechanical contribution of nucleus in adherent cells to bearing intracellular stresses remains unclear. In this paper, the effects of fluid shear stress on morphology and elastic properties of endothelial nuclei were investigated. The morphological observation suggested that the nuclei in the cytoplasm were being vertically compressed under static conditions, whereas they were elongated and more compressed with a fluid shear stress of 2 Pa (20 dyn/cm2) onto the cell. The elongated nuclei remained the shape even after they were isolated from the cells. The micropipette aspiration technique on the isolated nuclei revealed that the elastic modulus of elongated nuclei, 0.62+/-0.15 kPa (n=13, mean+/-SD), was significantly higher than that of control nuclei, 0.42+/-0.12 kPa (n=11), suggesting that the nuclei remodeled their structure due to the shear stress. Based of these results and a transmission electron microscopy, a possibility of the nucleus as an intracellular compression-bearing organelle was proposed, which will impact interpretation of stress distribution in adherent cells.