Carmela Rianna

Topographic cell instructive patterns to control cell adhesion, polarization and migration

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.

Reversible Holographic Patterns on Azopolymers for Guiding Cell Adhesion and Orientation

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nanopatterning techniques have been employed to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated the response of NIH-3T3 cells to reversible topographic signals encoded on light-responsive azopolymer films. Switchable patterns were fabricated by means of a well-established holographic setup. Surface relief gratings were realized with Lloyds mirror system and erased with circularly polarized or incoherent light. Cell cytoskeleton organization and focal adhesion assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as a trigger. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way toward an in situ and real-time investigation of the material-cytoskeleton crosstalk caused by the intrinsic properties of azopolymers.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Cell mechanics as a marker for diseases: Biomedical applications of AFM

Many diseases are related to changes in cell mechanics. Atomic Force Microscopy (AFM) is one of the most suitable techniques allowing the investigation of both topography and mechanical properties of adherent cells with high spatial resolution under physiological conditions. Over the years the use of this technique in medical and clinical applications has largely increased, resulting in the notion of cell mechanics as a biomarker to discriminate between different physiological and pathological states of cells. Cell mechanics has proven to be a biophysical fingerprint able discerning between cell phenotypes, unraveling processes in aging or diseases, or even detecting and diagnosing cellular pathologies. We will review in this report some of the works on cell mechanics investigated by AFM with clinical and medical relevance in order to clarify the state of research in this field and to highlight the role of cell mechanics in the study of pathologies, focusing on cancer, blood and cardiovascular diseases.At...

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Measuring the viscoelastic creep of soft samples by step response AFM

We have measured the creep response of soft gels and cells after applying a step in loading force with atomic force microscopy (AFM). By analysing the creep response data using the standard linear solid model, we can quantify the viscous and elastic properties of these soft samples independently. Cells, in comparison with gels of similar softness, are much more viscous, as has been qualitatively observed in conventional force curve data before. Here, we quantify the spring constant and the viscous damping coefficient from the creep response data. We propose two different modes for applying a force step: (1) indirectly by increasing the sample height or (2) directly by employing magnetic cantilevers. Both lead to similar results, whereas the latter seems to be better defined since it resembles closely a constant strain mode. The former is easier to implement in most instruments, and thus may be preferable from a practical point of view. Creep analysis by step response is much more appropriate to analyse the viscoelastic response of soft samples like cells than the usually used force curve analysis.

The role of the microenvironment in the biophysics of cancer

During the last decades, cell mechanics has been recognized as a quantitative measure to discriminate between many physiological and pathological states of single cells. In the field of biophysics of cancer, a large body of research has been focused on the comparison between normal and cancer mechanics and slowly the hypothesis that cancer cells are softer than their normal counterparts has been accepted, even though in situ tumor tissue is usually stiffer than the surrounding normal tissue. This corroborates the idea that the extra-cellular matrix (ECM) has a critical role in regulating tumor cell properties and behavior. Rearrangements in ECM can lead to changes in cancer cell mechanics and in specific conditions the general assumption about cancer cell softening could be confuted. Here, we highlight the contribution of ECM in cancer cell mechanics and argue that the statement that cancer cells are softer than normal cells should be firmly related to the properties of cell environment and the specific stage of cancer cell progression. In particular, we will discuss that when employing cell mechanics in cancer diagnosis and discrimination, the chemical, the topographical and - last but not least - the mechanical properties of the microenvironment are very important.

Passive microrheology of normal and cancer cells after ML7 treatment by atomic force microscopy

Mechanical properties of living cancer and normal thyroidal cells were investigated by atomic force microscopy (AFM). Cell mechanics was compared before and after treatment with ML7, which is known to reduce myosin activity and induce softening of cell structures. We recorded force curves with extended dwell time of 6 seconds in contact at maximum forces from 500 pN to 1 nN. Data were analyzed within different frameworks: Hertz fit was applied in order to evaluate differences in Young’s moduli among cell types and conditions, while the fluctuations of the cantilever in contact with cells were analyzed with both conventional algorithms (probability density function and power spectral density) and multifractal detrended fluctuation analysis (MF-DFA). We found that cancer cells were softer than normal cells and ML7 had a substantial softening effect on normal cells, but only a marginal one on cancer cells. Moreover, we observed that all recorded signals for normal and cancer cells were monofractal with small...

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts

Mechanical properties of myofibroblasts play a key role in Dupuytrens disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytrens nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor‐β1 (TGF‐β1) is known to induce expression of α–smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF‐β1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytrens fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytrens, normal and scar fibroblasts, respectively; (2) TGF‐β1 enhances the level of α–smooth muscle actin expression and thus cell stiffness in Dupuytrens fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytrens fibroblasts; and (4) Dupuytrens fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

Imaging and characterization of surface relief gratings on azopolymer by digital holographic microscopy

Azopolymer materials belong to family special materials, which are subject to photo-isomerization when illuminated by appropriate light wavelength. Optical characterization of azopolymer materials is interesting because they can be patterned when illuminated by coherent polarized light with potentially interesting applications in the biotechnology, photonic elements, molding templates, etch masks and micro-nanochannels. The interference lithography is an excellent tool to trigger the isomerization reaction on the material. During this work, switchable patterns were fabricated by means of a well established holographic set-up: surface relief gratings (SRGs) were realized with Lloyd’s mirror system. Moreover, optical characterization of the material was performed, starting from a commercial one and using a new way to analyse SRGs by means of Digital Holography Microscopy, to determine relevant parameters for the realization of the patterns with different shape and size. Some preliminary results of the influence of such patterns on the cell behavior were shown.