1 Introduction

Europe faces crucial health challenges due to demographic aging, an increase of sedentary- and nutrition-linked problems and the emergence of infectious diseases. The population of Europeans aged 65 and more is expected to double over the next 50 years, with the subsequent increase of chronic diseases, including age-related comorbidities, like neurodegenerative diseases and cancer. Early detection and diagnosis of diseases provide essential tools for a better quality of life for aged people by allowing improved prognosis and personalized healthcare to optimize therapeutic strategies.

Nanotechnology allows the manipulation and inspection of matter at the nanometer scale with unprecedented sensitivity and spatial resolution, also providing new perspectives for the investigation of biological systems. The nanoscale investigation of cells and tissues revealed that specific diseases have well-pronounced mechanical fingerprints. For example, cancerous cells have shown to be typically significantly softer than normal cells, while the extracellular matrix (ECM) of cancerous tissue is typically stiffer than in normal cases. It is not surprising since anatomopathologists and histologists in the clinics are used to observe and often to sense directly by palpation, significant mechanical changes occurring in diseased tissues and organs as a consequence of the progression of the pathology.

Several nanoscale experimental techniques such as atomic force microscopy (AFM), nanoindenters, magnetic or optical tweezers, super-resolution photonic microscopy, the use of micropatterns and micropillars, and microfluidics have allowed to reliably probe the mechanical properties of cells and tissues within the research conducted at the laboratory level. Quantitative cell and tissue (nano)mechanobiology offer the possibility of adding to the growing list of molecular markers a new class of markers based on physical properties, specifically on suitable elastic and viscoelastic parameters (a short historical perspective on mechanobiology is presented at the end of this section). Therefore, it is appropriate to say that a new paradigm arose - the use of the mechanical fingerprint of cells and tissues to detect and diagnose diseases. Crucial, for this purpose, is paving the road to bring nanomechanical tests to the clinic.

Among several approaches that have been developed, AFM presents some unique advantages.

It combines topographical information with force mapping allowing to measure adhesive and mechanical (rheological) properties of cells and tissues. In addition, when using functionalized probes, specific molecular interactions can be probed with high spatial and force resolution. On the other hand, it is fair to note that AFM is still not popularized within the biology and medical community and is not yet included in the toolbox of cell mechanobiologists. Despite the reported successes of AFM in the study of biological systems and its potentialities, still to be fully developed in the research milieu, the mechanical phenotyping of clinically relevant samples aimed at producing diagnostic cues requires novel, clinic-oriented tools, featuring ease of use and high throughput, which are still to be developed, although interesting products are appearing on the market. At the same time, the standardization of procedures for both preparation and mechanical testing of clinical samples must be implemented; when framed into the clinical environment, these objectives represent far greater challenges compared to when they are pursued among different research laboratories, an already complex task that has been only partially fulfilled to date.

Given that innovation in the health field proceeds through the development of advanced knowledge, novel methodologies and technologies, and their application to human diseases in the places where diseases are confronted, the clinics, the tools to support this challenging strategy, are interdisciplinary and intersectoral research, cooperation through networking, and effective communication and sharing of methodologies. A recently funded EU project, Phys2BioMed,1 aims at accomplishing these tasks through the interdisciplinary and cross-sectoral training of a team of early-stage researchers, taking advantage of a network of research institutions, companies, and hospitals.

This book is aimed at collecting and summarizing, to the primary benefit of young researchers but also of senior scientists involved in interdisciplinary studies, the present knowledge on the fundamental biomechanical markers of diseases, like cancer, and on the experimental techniques that are used to characterize the mechanical properties of cells and tissues; we emphasized the application of these techniques to the study of clinically relevant samples.

1.1 History of Biomechanical Investigations of Cells and Tissues

Or The Long Route from Biomechanics via Mechanobiology to Mechanics in Diseases

Biomechanics, that is, employing principles from mechanics to describe biological systems has a very long tradition. The possibly first, clear, and prominent example was the physics behind blood circulation. Even the concept that blood circulates in the first place was, a (then controversially discussed) consequence of the simple fact that the heart is pumping more than 200 kg of blood per hour, raised by Harvey (1628). The shear amount suggests that there has to be a circular network, which at that time was not apparent, since the connection between the arterial and the venous system could not be seen: capillaries are just too small to be detected by the naked eye and optical microscopy was in its infancy then. This controversy could only be solved once optical microscopy became available. This early example shows how important the connection between developing new concepts and the availability of appropriate techniques is. Harvey can be considered as a disciple of Galileo, who is well known for his contributions to astronomy, but actually started studying medicine (Fung 1993) or, as we would call it nowadays, physiology. Other early examples of biomechanics are the work of Young (1800), understanding the generation of sound, which relies strongly on the elastic properties of the vocal cords, or understanding the function of the lung, which is an interplay of the mechanical properties of lung tissue (most importantly its very high extensibility and the hydrodynamics of airflow). Another more modern example is the biomechanics of our locomotor system, that is, the functional interplay of muscles, tendons, and the skeleton. This - rather macroscopic - application of mechanics in biology can be understood as applying the laws of mechanics, as a part of physics, and the concepts from engineering (envisioning the heart as a pump with valves, describing our locomotor system as a combination of forces and levers) to better understand how biological systems work.

To get a better insight into how mechanical aspects of biological systems are actually generated or created a more microscopic or even molecular understanding of mechanics in biology is needed. This more modern interpretation of the theme, which now is called mechanobiology, describes how active molecules (like the motor enzymes myosin or flagella in single-celled organisms) or structural molecules (like the actin cytoskeleton or polymeric proteins in the ECM) organize themselves in cells or tissues, generate forces, or are affected by external forces (deform) or even sense external forces (mechanosensation). These modern concepts require the availability of microscopic and structural techniques to characterize tissues, cells, and macromolecules like polymers down to nanometer dimensions. It also requires tools to sense and apply mechanical forces at increasingly smaller length and force scales.

Thus, the field profited and progressed from various techniques, which became available to study mechanical properties of cells and tissues. To highlight this idea (not giving a concise overview here), we just present key pioneering concepts here: (1) micropipette aspiration (Hochmuth 2000), first developed by Hochmuth (1993) to characterize red blood cells and later applied by Evans (1989) to measure the membrane properties of granulocytes. This technique, which was the first to probe viscoelastic properties of cells, and thus is conceptionally very important. However, its applicability was limited since it could not apply to adherent cells and tissues. (2) Scanning acoustic microscopy overcomes these limitations since it could probe mechanical properties of three-dimensional samples of cells and tissues, as shown by Bereiter-Hahn and his group (Kundu 1991). However, since the shear modulus has been derived from the speed of sound, which mainly reflects the properties of water, only affected to some degree by the polymeric network within and between cells, this technique has not found widespread applications. (3) Finally, the cell poker, developed by Eliot Elson...