Cellular Mechanotransduction: From Tension to Function

Living cells are constantly exposed to mechanical stimuli arising from the surrounding extracellular matrix (ECM) or from neighboring cells. The intracellular molecular processes through which such physical cues are transformed into a biological response are collectively dubbed as mechanotransduction and are of fundamental importance to help the cell timely adapt to the continuous dynamic modifications of the microenvironment. Local changes in ECM composition and mechanics are driven by a feed forward interplay between the cell and the matrix itself, with the first depositing ECM proteins that in turn will impact on the surrounding cells. As such, these changes occur regularly during tissue development and are a hallmark of the pathologies of aging. Only lately, though, the importance of mechanical cues in controlling cell function (e.g., proliferation, differentiation, migration) has been acknowledged. Here we provide a critical review of the recent insights into the molecular basis of cellular mechanotransduction, by analyzing how mechanical stimuli get transformed into a given biological response through the activation of a peculiar genetic program. Specifically, by recapitulating the processes involved in the interpretation of ECM remodeling by Focal Adhesions at cell-matrix interphase, we revise the role of cytoskeleton tension as the second messenger of the mechanotransduction process and the action of mechano-responsive shuttling proteins converging on stage and cell-specific transcription factors. Finally, we give few paradigmatic examples highlighting the emerging role of malfunctions in cell mechanosensing apparatus in the onset and progression of pathologies

DEFORMATION RESPONSE OF POLYDIMETHYLSILOXANE SUBSTRATES SUBJECTED TO UNIAXIAL QUASI-STATIC LOADING

To investigate cellular response of cardiomyocytes to substrate mechanics, biocompatible material with stiffness in physiological range is needed. PDMS based material is used for construction of microfluidic organ on chip devices for cell culture due to ease of device preparation, bonding, and possibility of surface functionalization. However it has stiffness orders of magnitude out of physiological range. Therefore, we adapted recently available protocol aiming to prepare substrates which offer stiffness in physiological range 5−100kPa using various mixtures of Sylgard. An in-house developed loading device with single micron position tracking accuracy and sub-micron position sensitivity was adapted for this experimental campaign. All batches of the samples were subjected to uniaxial loading. During quasi-static experiment the samples were compressed to minimally 40% deformation. The results are represented in the form of stress-strain curves calculated from the acquired force and displacement data and elastic moduli are estimated

Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and Microengineering

Recent advances in biomedical technologies are mostly related to the convergence of biology with microengineering. For instance, microfluidic devices are now commonly found in most research centers, clinics and hospitals, contributing to more accurate studies and therapies as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most remarkably, integration of cellularized constructs within microengineered platforms has enabled the recapitulation of the physiological and pathological conditions of complex tissues and organs. The so-called organ-on-a-chip technology, which represents a new avenue in the field of advanced in vitro models, with the potential to revolutionize current approaches to drug screening and toxicology studies. This review aims to highlight recent advances of microfluidic-based devices towards a body-on-a-chip concept, exploring their technology and broad applications in the biomedical field.European Regional Development Fund-Project FNUSA-ICRC [CZ.1.05/1.1.00/02.0123]; Fundacao para a Ciencia e a Tecnologia (FCT), Portugal [UID/BIM/04773/2013]; Internal Research Grant Program, Universita Campus Bio-Medico di Romainfo:eu-repo/semantics/publishedVersio

Advanced and Rationalized Atomic Force Microscopy Analysis Unveils Specific Properties of Controlled Cell Mechanics

The cell biomechanical properties play a key role in the determination of the changes during the essential cellular functions, such as contraction, growth, and migration. Recent advances in nano-technologies have enabled the development of new experimental and modeling approaches to study cell biomechanics, with a level of insights and reliability that were not possible in the past. The use of atomic force microscopy (AFM) for force spectroscopy allows nanoscale mapping of the cell topography and mechanical properties under, nearly physiological conditions. A proper evaluation process of such data is an essential factor to obtain accurate values of the cell elastic properties (primarily Young's modulus). Several numerical models were published in the literature, describing the depth sensing indentation as interaction process between the elastic surface and indenting probe. However, many studies are still relying on the nowadays outdated Hertzian model from the nineteenth century, or its modification by Sneddon. The lack of comparison between the Hertz/Sneddon model with their modern modifications blocks the development of advanced analysis software and further progress of AFM promising technology into biological sciences. In this work, we applied a rationalized use of mechanical models for advanced postprocessing and interpretation of AFM data. We investigated the effect of the mechanical model choice on the final evaluation of cellular elasticity. We then selected samples subjected to different physicochemical modulators, to show how a critical use of AFM data handling can provide more information than simple elastic modulus estimation. Our contribution is intended as a methodological discussion of the limitations and benefits of AFM-based advanced mechanical analysis, to refine the quantification of cellular elastic properties and its correlation to undergoing cellular processes in vitro

Dissecting the intracellular signalling and fate of a DNA nanosensor by super-resolution and quantitative microscopy

DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-kappa B molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu

Anthropometric evaluation in older individuals in relation to their physical activity level

Individuals older than 55 years tend to adopt sedentary lifestyles (European Commission, 2014) and unhealthy eating attitudes (Ahmed and Haboubi, 2010), which lead to an increase of body fat and risks of non-communicable diseases (Rosembloom and Bahnes, 2006; Sallinen et al., 2008). To investigate the association between the level of involvement in physical activity (PA) on anthropometric features with advancing age, height, body mass, waist (WC) and hip (HC) circumference, and triceps skinfold were measured in 20 athletes (training: >5 hr.week-1), 37 physically active (structured PA: 2 hr.week-1), and 42 sedentary (<1 hr.week-1), older individuals (55-84 yrs). Moreover body mass index (BMI), waist-hip (WHi) and waist-height (WHe) ratio, and fat arm index were calculated. A 2 (gender) x 3 (activity level) verified differences (p<0.05) between groups. No difference for gender emerged for BMI, HC, and WHi. Athletes showed lower (p<0.05) BMI, WC, HC, WHi, triceps skinfold, and arm fat index, and higher height values than physically active and sedentary counterparts. For body mass, a difference (p<0.05) emerged only between athletes with respect to sedentary counterpart. No differences emerged between physically active and sedentary groups. Findings indicate that in older ages only a high physical activity level allows controlling the anthropometric features, thus posing senior athletes at a lower risk of related non-communicable diseases

INFLUENCE OF PRINTING AND LOADING DIRECTION ON MECHANICAL RESPONSE IN 3D PRINTED MODELS OF HUMAN TRABECULAR BONE

The paper deals with investigation on directional variations of mechanical response in 3D printed models of human trabecular bone. Sample of trabecular bone tissue was resected from human donor and 3D model was obtained by X-ray computed tomography. Then a series of cubical samples was prepared by additive manufacturing technique and tested by uniaxial compression loading mode. Mechanical response was compared in nine different combinations of direction of 3D printing and loading direction. The results show neglectible influence on the deformation response in elastic region (stiffness) and significant changes of the behaviour in plastic region (stress and strain at yield point, strain at full collapse)

Cardiac fibroblasts and mechanosensation in heart development, health and disease

The term 'mechanosensation' describes the capacity of cells to translate mechanical stimuli into the coordinated regulation of intracellular signals, cellular function, gene expression and epigenetic programming. This capacity is related not only to the sensitivity of the cells to tissue motion, but also to the decryption of tissue geometric arrangement and mechanical properties. The cardiac stroma, composed of fibroblasts, has been historically considered a mechanically passive component of the heart. However, the latest research suggests that the mechanical functions of these cells are an active and necessary component of the developmental biology programme of the heart that is involved in myocardial growth and homeostasis, and a crucial determinant of cardiac repair and disease. In this Review, we discuss the general concept of cell mechanosensation and force generation as potent regulators in heart development and pathology, and describe the integration of mechanical and biohumoral pathways predisposing the heart to fibrosis and failure. Next, we address the use of 3D culture systems to integrate tissue mechanics to mimic cardiac remodelling. Finally, we highlight the potential of mechanotherapeutic strategies, including pharmacological treatment and device-mediated left ventricular unloading, to reverse remodelling in the failing heart