[Experimental studies of the biomechanical properties of the cornea]. / Eksperimental'nye issledovaniya biomekhanicheskikh svoistv rogovitsy.

The review presents the results of experimental studies of the biomechanical properties of the cornea. Selective evaluation of the individual corneal structures (for example, limiting membranes) using classical mechanical tests is to a certain extent limited due to the rather small thickness of these structures and the related difficulties in sample fixation. In real practice, the use of a method better adapted for conducting such studies - atomic force microscopy (AFM) - remains promising, since on the one hand it eliminates the need for mechanical capture and retention of the sample, and on the other - provides the capability for studying its segments separately.

Role of actin-binding proteins in the regulation of cellular mechanics.

The viscoelastic parameters of the cell can report on the cell state, cellular processes and diseases. Cell mechanics strongly rely on the properties of the cytoskeleton, an important system of subcellular filaments, especially on the high-level structures that actin forms together with actin-binding proteins (ABPs). In normal cells, components of the cytoskeleton are highly integrated, and their functions are well orchestrated. In contrast, impaired expression and functioning of ABPs lead to the increasing ability of cancer cells to resist chemotherapy and metastasize. ABP-mediated changes in the cytoskeleton architecture can lead to changes in the mechanical properties of the actin network, both locally and at the level of the whole cell. Until now, in cancer-related studies, mechanical data have been used less frequently, compared to biochemical tests or cell migration assays. Here, we will review current methods for analyzing the mechanical properties of cells and provide the available data on the contribution of ABPs in determining cell mechanical properties important for the investigation of cellular functions, particularly in cancers.

Viscoelasticity in simple indentation-cycle experiments: a computational study.

Instrumented indentation has become an indispensable tool for quantitative analysis of the mechanical properties of soft polymers and biological samples at different length scales. These types of samples are known for their prominent viscoelastic behavior, and attempts to calculate such properties from the indentation data are constantly made. The simplest indentation experiment presents a cycle of approach (deepening into the sample) and retraction of the indenter, with the output of the force and indentation depth as functions of time and a force versus indentation dependency (force curve). The linear viscoelastic theory based on the elastic-viscoelastic correspondence principle might predict the shape of force curves based on the experimental conditions and underlying relaxation function of the sample. Here, we conducted a computational analysis based on this theory and studied how the force curves were affected by the indenter geometry, type of indentation (triangular or sinusoidal ramp), and the relaxation functions. The relaxation functions of both traditional and fractional viscoelastic models were considered. The curves obtained from the analytical solutions, numerical algorithm and finite element simulations matched each other well. Common trends for the curve-related parameters (apparent Young's modulus, normalized hysteresis area, and curve exponent) were revealed. Importantly, the apparent Young's modulus, obtained by fitting the approach curve to the elastic model, demonstrated a direct relation to the relaxation function for all the tested cases. The study will help researchers to verify which model is more appropriate for the sample description without extensive calculations from the basic curve parameters and their dependency on the indentation rate.

Biomechanical properties of the lens capsule: A review.

The lens capsule, a thin specialized basement membrane that encloses the crystalline lens, is essential for both the structural and biomechanical integrity of the lens. Knowing the mechanical properties of the lens capsule is important for understanding its physiological functioning, role in accommodation, age-related changes, and for providing a better treatment of a cataract. In this review, we have described the techniques used for the lens capsule biomechanical testing on the macro- and microscale and summarized the current knowledge about its mechanical properties.

Digging deeper: structural background of PEGylated fibrin gels in cell migration and lumenogenesis.

Fibrin is a well-known tool in tissue engineering, but the structure of its modifications created to improve its properties remains undiscussed despite its importance, e.g. in designing biomaterials that ensure cell migration and lumenogenesis. We sought to uncover the structural aspects of PEGylated fibrin hydrogels shown to contribute to angiogenesis. The analysis of the small-angle X-ray scattering (SAXS) data and ab initio modeling revealed that the PEGylation of fibrinogen led to the formation of oligomeric species, which are larger at a higher PEG : fibrinogen molar ratio. The improvement of optical properties was provided by the decrease in aggregates' sizes and also by retaining the bound water. Compared to the native fibrin, the structure of the 5 : 1 PEGylated fibrin gel consisted of homogenously distributed flexible fibrils with a smaller space between them. Moreover, as arginylglycylaspartic acid (RGD) sites may be partly bound to PEG-NHS or masked because of the oligomerization, the number of adhesion sites may be slightly reduced that may provide the better cell migration and formation of continuous capillary-like structures.

Viscoelastic mapping of cells based on fast force volume and PeakForce Tapping.

Development of fast force volume (FFV), PeakForce Tapping (PFT), and related AFM techniques allow fast acquisition and mapping of a sample's mechanical properties. The methods are well-suited for studying soft biological samples like living cells in a liquid environment. However, the question remains how the measured mechanical properties are related to those acquired with the classical force volume (FV) technique conducted at low indentation rates. The difference is coming mostly from the pronounced viscoelastic behavior of cells, making apparent elastic parameters depending on the probing rate. Here, the viscoelastic analysis was applied directly to the force curves acquired with force volume or PeakForce Tapping by their post-processing based on the Ting's model. Maps from classical force volume, FFV and PFT obtained using special PFT cantilevers and cantilevers modified with microspheres were compared here. With the correct viscoelastic model, which was found to be the power-law rheology model, all the techniques have provided self-consistent results. The techniques were further modified for the mapping of the viscoelastic model-independent complex Young's modulus.

Application of the Johnson-Kendall-Roberts model in AFM-based mechanical measurements on cells and gel.

The force-distance curves (FCs) obtained by the atomic force microscope (AFM) with colloid probes contain information about both the viscoelastic properties and adhesion of a sample. Here, we processed both the approach and retraction parts of FCs obtained on polyacrylamide gels (in water or PBS) and Vero cells (in a culture medium). The Johnson-Kendall-Roberts model was applied to the retraction curves to account for the adhesion. The effects of loading rate, holding time and indentation depth on adhesion force and Young's modulus, calculated from approach and retraction curves, were studied. It was shown that both bulk and local interfacial viscoelasticity can affect the observed approach-retraction hysteresis and measured parameters. The addition of 1% bovine serum albumin (BSA) decreased adhesion of the probe to the PAA gel surface, so interfacial viscoelasticity effects were diminished. On the contrary, the adhesiveness of Vero cells increased after BSA addition, indicating the complex nature of the cell-probe interaction.

Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells.

The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900 Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400 Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology.

Mechanical properties of fibroblasts depend on level of cancer transformation.

Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Young's modulus of cells. In all cases the Young's moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40-80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.

The effects of confluency on cell mechanical properties.

Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4-1.7 times) Young's modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Young's modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties.

Atomic force microscopy of living and fixed Xenopus laevis embryos.

Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.

Atomic force microscopy study of the arrangement and mechanical properties of astrocytic cytoskeleton in growth medium.

Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young's modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement inin vitromodels of neurodegeneration.