Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

University of Memphis

The mechanical response of soft tissue is commonly characterized from unconfined uniaxial compression experiments on cylindrical samples. However, friction between the sample and the compression platens is inevitable and hard to quantify. One alternative is to adhere the sample to the platens, which leads to a known no-slip boundary condition, but the resulting nonuniform state of stress in the sample makes it difficult to determine its material parameters. This paper presents an approach to extract the nonlinear material properties of soft tissue (such as liver) directly from no-slip experiments using a set of computationally determined correction factors. We assume that liver tissue is an isotropic, incompressible hyperelastic material characterized by the exponential form of strain energy function. The proposed approach is applied to data from experiments on bovine liver tissue. Results show that the apparent material properties, i.e., those determined from no-slip experiments ignoring the no-slip conditions, can differ from the true material properties by as much as 50% for the exponential material model. The proposed correction approach allows one to determine the true material parameters directly from no-slip experiments and can be easily extended to other forms of hyperelastic material models.

The nonlinear material properties of liver tissue determined from no-slip uniaxial compression experiments.

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy.

In the past decade, novel tools for surgical planning and disease diagnosis have been developed to detect the liver's mechanical properties. Some tools utilize superficial indentation type measurements to determine the elasticity of the liver parenchyma and to assume material homogeneity. In fact, the liver is a soft tissue covered with a connective sheathing that is called Glisson's capsule. This article quantifies the effect of this capsule on the measured or "effective" elastic modulus obtained by indentation with a spherical geometry. Two sets of parametric computational studies in which the Glisson capsule thickness and elasticity were varied, demonstrated the relationship between the measured elastic response and the underlying parenchymal elastic response. Previously reported in vivo indentation data on the human liver were utilized to determine the elasticity of its parenchyma. The results indicated a linear relationship between the effective (measured) elastic response and the underlying parenchyma for the Glisson capsule thicknesses considered. When previously published human liver indentation data were analyzed, the measured elastic modulus was nearly 6.9% greater than the underlying parenchyma elastic modulus. Although the analyzed data were obtained from a single liver and yet displayed a significant variation, the Glisson capsule elasticity induced a significant but systematic error as well. The Glisson capsule thickness error was negligible for capsule parameters associated with a normal liver. Based on this work, an emphasis on the Glisson capsule's contribution to the mechanical response of the liver would enhance the clinical potential of indentation-based novel tools for liver care.

The effect of Glisson's capsule on the superficial elasticity measurements of the liver.

The mechanical response of most soft tissue is considered to be viscohyperelastic, making the development of accurate constitutive models a challenging task. In this article, we present a constitutive model for bovine liver tissue that utilizes a viscous dissipation potential, and use it to model the response of bovine liver tissue at strain rates ranging from 0.001 to 0.04 s(-1). On the material modeling front of this study, the free energy is assumed to depend on the right Cauchy-Green deformation tensor, whereas a separate rate-dependent viscous potential is posited to characterize viscoelasticity. This viscous dissipation component is a function of the time rate of change of the right Cauchy-Green deformation tensor. On the experimental front, no-slip uniaxial compression experiments are conducted on bovine liver tissue at various strain rates. A numerical correction approach is used to account for the no-slip edge conditions, and the constitutive model is fit to the resulting corrected stress-strain data. The complete derivation of the material model, its implementation in the finite element software package ABAQUS, and a validation study are presented in this article. The results show that bovine liver tissue exhibits a strong strain-rate dependence even at the low strain rates considered here and that the proposed constitutive model is able to accurately describe this response.

Strain rate-dependent viscohyperelastic constitutive modeling of bovine liver tissue.

The pulmonary alveolus, terminal gas-exchange unit of the lung, is composed of alveolar epithelial and endothelial cells separated by a thin basement membrane and interstitial space. These cells participate in the maintenance of a delicate system regulated not only by biological factors but also by the mechanical environment of the lung, which undergoes dynamic deformation during breathing. Clinical and animal studies as well as cell culture studies point toward a strong influence of mechanical forces on lung cells and tissues including effects on growth and repair, surfactant release, injury, and inflammation. However, despite substantial advances in our understanding of lung mechanics over the last half century, there are still many unanswered questions regarding the micromechanics of the alveolus and how it deforms during lung inflation. Therefore, the aims of this review are to draw a multidisciplinary account of the mechanics of the alveolus on the basis of its structure, biology, and chemistry and to compare estimates of alveolar deformation from previous studies.

What do we know about mechanical strain in lung alveoli?

We investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)-Cre with Kif3a(flox/null) mice. Conditional Kif3a-null mice (Kif3a(Oc-cKO)) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3a(Oc-cKO) mice developed osteopenia by 6 weeks of age unlike Kif3a(flox/+) control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3a(flox/+) and Kif3a(flox/null) heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3a(Oc-cKO) mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3a(Oc-cKO) mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3a(Oc-cKO) mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated β-catenin and Axin2 expression were also attenuated in Kif3a(Oc-cKO) bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling.

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia.

Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.

Hyperoxia alters the mechanical properties of alveolar epithelial cells.

Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis.

Mechanobiology in lung epithelial cells: measurements, perturbations, and responses.

The use of modeling and simulation is growing rapidly in applications such as surgery simulations, injury mechanics and tissue engineering. The aim of this study is to model and simulate tissue tearing and the resulting failure for use in such applications. In particular, our goal is to characterize the mechanics of mode I tearing in thin soft materials. We use the cohesive zone modeling approach to characterize the propagation of tears in a processed meat product (PMP). The bulk response of the PMP is modeled with a hyperelastic material model and the interface with a cohesive zone model. A multistep parameter estimation approach is developed to determine the bulk and the cohesive model parameters from uniaxial extension and tearing experiments. Results show that the proposed approach is able to capture both material and geometrical nonlinearities inherent to such problems, and accurately model the overall force-displacement response of thin soft materials during tearing at slow rates.

Cohesive zone modeling of mode I tearing in thin soft materials.

Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and while both hyperoxia and mechanical ventilation are necessary, each can independently cause additional injury. However, the precise mechanisms that lead to injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be stiffer due to increased filamentous actin (f-actin) formation via the GTPase RhoA and its effecter Rho kinase (ROCK). We examined cytoskeletal structures in cultured murine lung alveolar epithelial cells (MLE-12) under normoxic and hyperoxic (48 h) conditions. We also measured cell elasticity (E) using an atomic force microscope in the indenter mode. Hyperoxia caused increased f-actin stress fibers and bundle formation, an increase in g- and f-actin, an increase in nuclear area and a decrease in nuclear height, and cells became stiffer (higher E). Treatment with an inhibitor (Y-27632) of ROCK significantly decreased E and prevented the cytoskeletal changes, while it did not influence the nuclear height and area. Pre-exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched cyclically, but the ROCK inhibitor prevented this effect. Hyperoxia caused thickening of vinculin focal adhesion plaques, and inhibition of ROCK reduced the formation of distinct focal adhesion plaques. Phosphorylation of focal adhesion kinase was significantly reduced by both hyperoxia and treatment with Y-27632. Hyperoxia caused increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK.

Hyperoxia increases the elastic modulus of alveolar epithelial cells through Rho kinase.

Acute Respiratory Distress Syndrome remains challenging partially because the underlying mechanisms are poorly understood. While inflammation and loss of barrier function are associated with disease progression, our understanding of the biophysical mechanisms associated with ventilator-associated lung injury is incomplete. In this line of thinking, we recently showed that changes in the F-actin content and deformability of AECs lead to cell detachment with mechanical stretch. Elsewhere, we discovered that cytokine secretion and proliferation were regulated in part by the stretch-activated 2-pore domain K(+) (K2P) channel TREK-1 in alveolar epithelial cells (AECs). As such, the aim of the current study was to determine whether TREK-1 regulated the mechanobiology of AECs through cytoskeletal remodeling and cell detachment. Using a TREK-1-deficient human AEC line (A549), we examined the cytoskeleton by confocal microscopy and quantified differences in the F-actin content. We used nano-indentation with an atomic force microscope to measure the deformability of cells and detachment assays to quantify the level of injury in our monolayers. We found a decrease in F-actin and an increase in deformability in TREK-1 deficient cells compared to control cells. Although total vinculin and focal adhesion kinase (FAK) levels remained unchanged, focal adhesions appeared to be less prominent and phosphorylation of FAK at the Tyr(925) residue was greater in TREK-1 deficient cells. TREK-1 deficient cells have less F-actin and are more deformable making them more resistant to stretch-induced injury.

The 2-pore domain potassium channel TREK-1 regulates stretch-induced detachment of alveolar epithelial cells.

We examined the bone properties of BXD recombinant inbred (RI) mice by analyzing femur and tibia and compared their phenotypes of different compartments. 46 BXD RI mouse strains were analyzed including progenitor C57BL/6J (n = 16) and DBA/2J (n = 15) and two first filial generations (D2B6F1 and B6D2F1). Strain differences were observed in bone quality and structural properties (P < 0.05) in each bone profile (whole bone, cortical bone, or trabecular bone). It is well known that skeletal phenotypes are largely affected by genetic determinants and genders, such as bone mineral density (BMD). While genetics and gender appear expectedly as the major determinants of bone mass and structure, significant correlations were also observed between femur and tibia. More importantly, positive and negative femur-tibia associations indicated that genetic makeup had an influence on skeletal integrity. We conclude that (a) femur-tibia association in bone morphological properties significantly varies from strain to strain, which may be caused by genetic differences among strains, and (b) strainwise variations were seen in bone mass, bone morphology, and bone microarchitecture along with bone structural property.

Bone morphology in 46 BXD recombinant inbred strains and femur-tibia correlation.

The physis, or growth plate, is a complex disc-shaped cartilage structure that is responsible for longitudinal bone growth. In this study, a multi-scale computational approach was undertaken to better understand how physiological loads are experienced by chondrocytes embedded inside chondrons when subjected to moderate strain under instantaneous compressive loading of the growth plate. Models of representative samples of compressed bone/growth-plate/bone from a 0.67 mm thick 4-month old bovine proximal tibial physis were subjected to a prescribed displacement equal to 20% of the growth plate thickness. At the macroscale level, the applied compressive deformation resulted in an overall compressive strain across the proliferative-hypertrophic zone of 17%. The microscale model predicted that chondrocytes sustained compressive height strains of 12% and 6% in the proliferative and hypertrophic zones, respectively, in the interior regions of the plate. This pattern was reversed within the outer 300 μm region at the free surface where cells were compressed by 10% in the proliferative and 26% in the hypertrophic zones, in agreement with experimental observations. This work provides a new approach to study growth plate behavior under compression and illustrates the need for combining computational and experimental methods to better understand the chondrocyte mechanics in the growth plate cartilage. While the current model is relevant to fast dynamic events, such as heel strike in walking, we believe this approach provides new insight into the mechanical factors that regulate bone growth at the cell level and provides a basis for developing models to help interpret experimental results at varying time scales.

Regional variations in growth plate chondrocyte deformation as predicted by three-dimensional multi-scale simulations.

TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.

Esra Roan

Department of Biomedical Engineering

University of Memphis

Live Cell Imaging during Mechanical Stretch

Esra Roan

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University of Memphis

Live Cell Imaging during Mechanical Stretch

Department of Biomedical Engineering