Chapter 1: An Introduction to Material-based Mechanobiology

“The force will be with you, always,” a famous quote by master Yoda in the popular fiction film Star Wars, applies to our real lives. Forces are involved not only in responses within sensory tissues but also in any part of our body. The movement of skeletal muscles and organs, blood flow, and gravity generate mechanical stimuli, and cells use these mechanical cues to regulate their morphology, proliferation, migration, and functions in order to eventually control various physiological and pathological processes, such as development and cancer progression.^1–3  Conventional molecular biological studies have not paid attention to, or rather had forgotten (see Section 1.2), the role of these mechanical forces. However, the discovery of various mechanosensory molecules responsible for cellular force perception has led to increased research on the roles of biological forces over time. After the initial identification of mechanosensitive ion channels existing at the interface between the cell and the outside world, many new mechanosensing machineries have been found, such as molecules involved in focal adhesion, cytoskeleton structures, and even nuclei as intracellular organelles. All such mechanosensing machineries perceive external forces and translate them into “molecular words” to regulate signal transduction and gene expression. At the same time, cells exert forces on substrates and adjacent cells via the functions of actin cytoskeleton and adhesion machineries and utilise them to sense the mechanical properties of their surroundings and dictate their activities and fates. Moreover, the forces exerted among neighbouring cells or those between cells and connective tissues or body fluids do not stay constant; rather, they are constantly fluctuating and play essential roles in time-evolving biological processes, including tissue formation, wound healing, and the development and metastasis of cancer. Another important feature is that forces can directly react with the multilayered architectures of living systems, ranging from nanoscale proteins, subcellular organelles, cells, tissues, and organs to whole bodies; we could coin the hierarchical nature as “mechanoarchitectonics”. Therefore, it is necessary to develop new methodologies for quantitatively sensing the forces in real time or, the other way around, for loading time-evolving “dynamic” forces in various spatiotemporal scales. Such technologies will elucidate a complete picture of the relationships of forces in our body and eventually create a new paradigm in biology and medicine. However, the measurement and manipulation of these invisible forces are challenging for most molecular biologists, whose main targets have been the “molecular words”. On the other hand, material scientists are more familiar with the interactions and transmission of forces with and through materials, which can be used as translators of forces in multiple spatiotemporal scales. This compatibility has led to a new discipline in mechanobiology studies, based on engineered materials, whose aim is to measure and manipulate molecular and cellular forces, wherein materials play an essential role. This direction of research can be called “material-based mechanobiology”, which this book addresses. Mechanobiology research is heading toward the peak of its success due to the availability of new biological tools. The last two decades have envisioned the exponential growth of material-based methodologies, which has motivated us to collect a wide variety of work from the front-runners of this field. In this chapter, we will introduce the origin and a brief history of mechanobiology research in general, followed by depicting the developmental processes of material-based mechanobiology, whose details will be introduced in the following chapters.

The origin of mechanobiology goes back to the late 19th century when the German surgeon Julius Wolff hypothesised that bones had evolved to maintain mechanically optimal geometries with maximal strength and minimal weight. This concept later came to be known as Wolff's law of bone remodelling.⁴  In 1917, D'Arcy Thompson, a biologist, published a book titled On Growth and Form, which describes the logic of physics and mathematics that can be applied to explain structure formation and the morphogenesis of plants and animals. Furthermore, there is a clear link between the morphology of living organisms and the mechanical forces they encounter. The age before DNA discovery and the emergence of molecular biology had no molecular insights and hence was discussed only from the mathematical and physical viewpoints.

In the 1950s, researchers started investigating the domain of cellular scale due to the establishment of cell culture technologies around that time. It was reported that cancer cells, unlike normal cells, maintain proliferative potential under nonadherent conditions such as on soft agar.^5,6  A separate study also showed that adding ionic drugs to erythrocytes caused morphological changes through changes in membrane tension.⁷  These facts suggest that the cells can mechanically sense extracellular matrix (ECM) cues and the membrane tension. Further research to uncover these mechanisms led to the discovery of focal adhesion, where the protein complexes at the cell–ECM interface act as bridges between the ECM and the actomyosin machinery to regulate cell adhesion and migration.

Meanwhile, muscle biophysicists have elucidated the essential role of actomyosin in muscle contraction.^8,9  The findings from these studies indicate that the actomyosin machinery-driven mechanical force strongly contributes not only to muscle contraction but also to cell division and migration. In particular, the actomyosin machinery in nonmuscle cells is coupled to cell–matrix adhesion sites. Therefore, it is reasonable to infer that the cells exert forces on the substrate using intrinsic contractility. However, this cellular force is too weak and could not be detected by the conventional tools available at that time. To tackle these technical challenges, Harris et al. fabricated a thin elastic silicone fluid substrate made of polydimethylsiloxane (PDMS), cross-linked only at the top (∼1 μm)by brief flaming, and they succeeded in visualising the traction forces generated by cells as a function of surface wrinkles on the cross-linked elastic substrate.¹⁰  This study showed for the first time that even nonmuscle fibroblasts generate forces on substrates, which led to the development of the next generation of tools for measuring cell traction (discussed in Chapters 3 and 4). Moreover, this study probably marked the first report of material science encounters in the field of mechanobiology.

Harris et al.'s report showed clear evidence that the cells are mechanically coupled to the ECM. However, the mechanisms with which these cells sense the mechanical properties of their surroundings still needed to be investigated. The first molecular evidence was obtained from analysing ion channels by patch-clamp recording (Figure 1.1A).¹¹  In this technique, the formation of tight sealing, called gigaseal, between the pipette and plasma membranes was critical to reduce the background noise.¹²  To achieve gigasealing, gentle suction was applied to the membrane before channel recording, which inevitably applied membrane tension to the patched membrane region. In 1984, Guharay et al. noticed single-channel conductance generation during this membrane sucking process and reported the presence of a mechanosensitive channel (MS channel), called a stretch-activated channel (Figure 1.1F);^13,14  ten years later, Sukharev et al. isolated the gene of an MS channel (MscL, MS channel of large conductance) from E. coli.¹⁵  This was the first study to identify molecular mechanosensors from nonsensory cells. At the same time, the discovery of the molecular identity of MS channels made molecular biological studies possible. A later study identified other MS channels, including TREK and Piezo channels, from eukaryotic cells. In particular, Piezo channels have been associated with human diseases related to mechanopathologies, hereditary xerocytosis, and generalised lymphatic dysplasia, thereby becoming important targets in mechanobiological studies.¹⁶ 

Meanwhile, the force transmission mechanisms at the cell–ECM interface have also been extensively investigated. In contrast to the research on membrane tension mechanosensing, where the patch-clamp technique provides a relatively simple way to investigate the forces involved, various experimental tools such as atomic force microscopy and optical/magnetic tweezers are needed for mechanobiology studies (Figure 1.1B,C).^17,18  In the 1970s, the existence of transmembrane proteins connecting the extracellular matrix (ECM) to the actin-based cytoskeleton began to be debated. Hynes and other groups had reported that extracellular fibronectin fibres were physically attached to the intracellular actin filaments, but their identity was not confirmed until the 1980s.^19–21  In 1986, this membrane protein complex was identified to be integrins.²²  Since then, at least 24 human integrin subtypes have been discovered. As most of the forces, including contractional forces, shear stresses, and hydrostatic pressures, result in stress concentration at the anchorage points at the cell–ECM interface, it is reasonable to hypothesise that integrins are involved in the mechanosensing process (Figure 1.1G).²³  Integrins exhibit several unique mechanoresponsive characteristics at the molecular level, including catch-bond and conformational changes or clustering.²⁴ 

In addition to integrins, another important intracellular mechanosensing molecule has been discovered in the focal adhesion regions. Talin is a protein associated with cytoskeleton (Figure 1.1H).^25–27  The N- and C-terminals of talin bind to integrins and actin cytoskeleton, respectively; thus the force from the actin cytoskeleton is also transmitted to talins. The talin rod domain has many binding sites for vinculin, most of which are hidden in the folded structure. Both in vitro and in vivo experiments have shown that the tensile force derived from the activity of myosin II in the cytoskeleton can cause them to unfold and elongate and expose the rod domain of the vinculin binding.^27,28  Therefore, such observations collectively indicate a mechanosensory role for talins.

Interestingly, actin cytoskeleton is not only the source of force generation through the activity of myosin, but it can also respond to forces by itself. More specifically, the degradation of actin filament can be observed upon relaxation of its tension,²⁹  which indicates its role as a mechanosensor against cellular tension (negative tension sensor).³⁰  The question then arises as to how does actin respond to such mechanical tension changes? In various actin-based intracellular architectures, such as bundles, lamellipodia, and cortex, actin filaments withstand mechanical loading against their global structures. This has been revealed by using diverse microscopy techniques, including electron microscopy, fluorescence resonance energy transfer (FRET), atomic force microscopy, and optical trapping (Figure 1.1B, C). The force application to the actin filament changes its three-dimensional structure³¹  thereby changing the affinity with various actin-binding proteins (ABPs).³⁰  Particularly, the tensile force increases the length of the actin filaments parallel to the direction of stretching, reducing the binding affinity to cofilin, an actin cleavage factor, increasing that to myosin II (Figure 1.1I),^29,32,33  and eventually stabilising the tensile fibres as the basis of various mechanical signalling processes.³⁴  On the contrary, the reduction of tension increases cofilin binding and decreases myosin binding; thereby the decrease in cellular tension promotes the degradation of actin filaments. Tension-induced changes in actin structural dynamics also affect the binding of actin nucleation proteins, such as Arp2/3.³⁵  Mechanically induced changes in actin-based structures also affect gene expression in at least some cell types, such as mesenchymal stem cell and breast cancer cell. As more tension fibres are formed during mechanical stimulation, the transcriptional conjugator YAP is translocated to the nucleus and activated. YAP is essential for Hippo signalling and mediates the increased expression of genes involved in cell proliferation and differentiation. Thus it is thought to result in physiological and pathophysiological processes due to the response of the actin cytoskeleton to extracellular mechanical forces.^36,37  In addition, the existence of the transcription conjugators in the downstream of actin-based signalling, including the Rho/Rho-kinase pathway, suggests triggering of gene expression regulation by mechanical forces.

In addition to cellular mechanosensing at the focal adhesion sites and cytoskeletal structures as previously mentioned, it has recently been shown that forces and deformations applied to the cellular surface can alter the shape of the nucleus via the force transmission through the cellular cytoskeleton. This nuclear deformation has a significant impact on the accessibility of the nuclear factors with the genetic codes in the nucleus. Therefore, the possibility of using cell nuclei as mechanosensors has been considered. Similar to the plasma membrane and cortex at the cell–ECM boundary, the nuclear envelope is a dynamic and force-sensitive interface between the cytoplasm and chromatin. The main structural cortex of the nuclear envelope is the lamina, composed of helix-rich fibrillar lamin proteins that assemble just below the inner nuclear membrane.³⁸  Two types of lamins, A-type (lamin A and lamin C) and B-type (lamin B1 and lamin B2), are tethered to the nucleus via the LINC complex, a linker of the nucleoskeleton and cytoskeleton.

Similarly, many mechanosensors have been identified and are still being sought, in different sizes and molecular assemblies, e.g. the cytoskeleton and nucleus, and their details are discussed in Chapter 2.

Two studies laid a strong foundation for material-based mechanobiology from an engineering perspective. One proposed the control of cell shape and the other modulation of the elastic modulus of the substrate. In parallel, materials for measuring forces applied at various scales and locations have been developed, and these have become indispensable technologies for mechanobiological research. In this section, we outline the development of material-based mechanobiology research in three primary directions: shape control, stiffness control, and force measurement, with a focus on the material aspect. In particular, cellular mechanoarchitectonics and the dynamic nature of mechanobiological responses will be discussed in wide variety of scale and time. Here, we first introduce these technologies for the control of cell form (geometry) and substrate mechanics, from which a wide variety of innovative materials and sciences have emerged.

As discussed in the previous section, the relationship between morphology and growth has been observed since the early stages of the history of mechanobiology. However, at that time, the central focus was on larger biological units, such as tissue and bones. In 1997, Chen et al. proved that a similar rule could be applied to single-cell growth.³⁹  They took advantage of microcontact printing based on PDMS elastomers to fabricate cell-adhesive islands in a nonadhesive background and demonstrated the relationship between the geometry of single cells and their growth. The cells switched from proliferative to apoptotic upon decreasing the spreading area, indicating that the shape of cells determines whether they live or die due to their local mechanical deformation. This success exemplified the usefulness of this fabrication technology for controlling cellular morphologies for mechanobiological studies. In microcontact printing technology, clean room environments are not needed after preparing the replica moulds, from which PDMS-based stamps can be produced repeatedly. Such user-friendly features are one of the biggest reasons for the widespread use of microcontact printing in mechanobiology studies.

Particularly, the development of soft lithography, including microcontact printing, after this period is attributed mainly to the characteristics of PDMS. PDMS is the most widely used silicone polymer due to the relatively lower-cost raw material. Furthermore, cross-linking is easy as it is a simple addition reaction in the presence of a catalyst. In addition, the glass transition temperature of PDMS is remarkably low (ca. −125 °C) among conventional polymers, and the cross-linked material is in a rubbery state at room temperature. The elastomeric feature of this material makes it easy to peel off from the master, and its high adhesiveness facilitates the fabrication of microfluidic devices that adhere to glass substrates. This easy processing technique enables its use in mechanobiology research, such as the development of micropillar devices for measuring cell traction force, cell response to shear stress from fluid flow and substrate stretching, cell manipulation using flow channels and stretch chamber, among others (Figure 1.1D).^40,41 

Moreover, it is possible to apply an elastic force to cells using its elastic properties directly. In addition, PDMS has high oxygen permeability owing to its large free volume, which enables oxygen supply to cells through the substrate when used as a scaffold material, thereby facilitating the culturing of iPS and ES cells.⁴²  This wide variety of mechanobiological research utilising the material characteristics of PDMS and the simplicity of soft lithography is described in more detail in Chapter 4.

On the other hand, a study focusing on cell shape showed that the control of geometrical cellular shape generates stress at the edge, where the active expression of lamellipodia is observed, which triggers cell migration and is associated with the regulation of biological activities by force.⁴³  A tensegrity model was proposed in which the actin skeleton was regarded as an elastic material and microtubules as rigid materials, and an explanation from the material mechanics point of view was attempted.^44,45  In addition to the effects of cell shape on proliferation, cell polarity, and migration, there have been gradual developments in the effects of cell differentiation,⁴⁶  cell collectives in 2D,⁴⁷  and 3D cell culture.⁴⁸  At the same time, new patterning techniques such as dip-pen nanolithography⁴⁹  and laser-based patterning⁵⁰  have been developed to improve accuracy and enable 3D modelling. Especially in 3D bioprinting of cells, inks are often required to have characteristics such as strain softening, so there was also a contribution to research on the development of such materials.^51,52  In addition, changes in intracellular stress distribution due to its shape, which are observed in single cells, can also be seen in cell clusters when cells are considered as a continuum model and in force-driven regioselective cell proliferation and phenotypic changes in tissues (such as epithelial–mesenchymal transition⁵³  and epigenetic factor reprogramming⁵⁴ ). These studies played a significant role in promoting the development of stem cell and cancer mechanobiology by recapitulating physiological 3D environments. More details on functional regulation by cell geometry are provided in Chapter 5.

On the other hand, attempts to utilise dynamic patterning are major progress in the field. As detailed in Chapter 6, materials that respond to heat, light, electric potential, etc., have been developed to control cell adhesion at the interface, the activity of adhesion ligands, and the highly specific click reaction. Such materials can be used to expand the cell adhesion area on demand, freely arrange multiple cells, and induce cell migration.⁵⁵  In particular, cell migration is a major issue in mechanobiology because the forces between cells and substrates are closely coupled to this phenomenon.⁵⁶  For example, after defining the stress distribution of cells and cell collectives, this method enabled mechanobiological investigation of the impact of initial geometry on the appearance of leader cells in subsequent migration behaviour, as well as the effect of the chemical and mechanical cues of the ECM on this behaviour, by defining the interfacial properties of functional materials.⁵⁷  Meanwhile, a new development of such dynamic materials is to utilise these materials to dynamically control the activity of ligands, rather than simply changing the geometry.⁵⁸  The reason behind such a development can be attributed to the dynamic change of force seen in the expansion and contraction of blood vessels in vivo. Moreover, ultrasound-based therapy has attracted much attention,⁵⁹  and it has recently been revealed that cells are more sensitive to the loading rate than to the equilibrium force.⁶⁰  The switchable interface is beneficial for dynamic mechanobiology research and leads to the development of materials with faster response times.

In addition to this approach of enlarging the size and dimension (e.g., from 2D to 3D) to resemble biological tissues and focus on temporal dynamics, research has also been conducted to downsize the controlling form to subcellular and molecular levels. This strategy leads to constraints on the arrangement and dynamics of molecules and molecular assemblies, and it allows us to explore mechanonanoarchitectonics, in which interactions at the molecular level are translated into cell size and cell populations. In particular, the block copolymer micellar nanolithography described in Chapter 7 is a technique for arranging gold nanoparticles in a hexagonal lattice based on the principle of colloidal lithography.⁶¹  The clustering behaviour of integrins can be controlled because these nanoparticles possess the size of a pair of integrin heterodimers, and the particle spacing can be precisely controlled according to the fabrication conditions. Their study demonstrated that integrins serve as a molecular ruler that can recognise the nanoscopic ligand spacings. Moreover, technological innovations such as applying substrates of various elastic moduli,⁶²  immobilisation of multiple ligands,⁶³  and the measurement of forces applied to molecules during endocytosis in combination with molecular force probes (Chapter 3) are also being investigated.

In the study of mechanobiology through geometric control using materials, it is clearly shown that controlling the cell shape and intracellular mechanical field is essential for the manifestation of cell functions. On the other hand, some evidence suggesting that the topography of the cell scaffold may influence cell polarisation and movement was reported in the early 20th century.⁶⁴  These results are not surprising considering that ECMs provide various topographies, such as fibre and groove structures, in vivo. However, those studies employed natural materials such as spider webs and fibrin, making it difficult to investigate the effects of topography on cell behaviour systematically. With the development of nano- and microfabrication technologies and material fabrication techniques, it has become possible to fabricate topographic substrates with various size and scale characteristics that mimic mechanoarchitecture in vivo, enabling us to study ECM topography's role in vitro. Synthetic topographic substrates can now be used to successfully replicate the contact guidance—the tendency of cells to shape and migrate along with topographic features and to create highly orientation-controlled cellular morphologies and tissue structures and functions, such as muscle and cardiac tissue found in vivo. Topography is currently being studied for its understanding as a major physical factor covered by mechanobiology research, similar to stiffness and geometry, and is discussed in detail in Chapter 8.

On the other hand, recent remarkable developments in electron microscopy and imaging techniques have contributed significantly to the multiscale structural analysis of ECM and ultrastructural analysis of tissues. Such developments have made it possible to design topographies based on biological principles. It is well known that changes in tissue stiffness accompany the development and progression of diseases such as cancer and fibrosis and that similar dynamic changes have been observed in ECM topography. To realise such dynamic topographic changes, it is difficult to use solely fabrication and material manufacturing technologies, as previously described; the development of new materials is required. If dynamic topography plays a role in pathological processes, as suggested by comparing ultrastructural analysis of normal and pathological tissues, this type of material could be a valuable tool for mechanobiology research and understanding physiological and pathological phenomena, including the development and progression of disease. Although this is still an emerging field, and the focus is on fundamental research on materials, such as materials development and the operating mechanism of variable topography, the mechanobiology research using materials with reconfigurable topography is described in detail in Chapter 9.

It is also known that cells sense the curvature of substrates larger than a single cell, leading to the alteration of their activities such as proliferation, migration, and differentiation, in contrast to the nanoscopic curvature and topography of surfaces smaller than that of a cell. Although this force perception should be closely interplayed with tissue formation, there has been no clear evidence of its effect until recently because the displacement is very small from the viewpoint of individual cells, combined with the technical difficulty of forming highly precise curved surfaces over a wide-range area. In particular, in mechanosensing for the nanoscopic curvature studied previously, it is reasonable to assume that molecules such as curvature sensing proteins and mechanosensitive (MS) ion channels, which are also involved in membrane trafficking systems, can sense the curvature. In contrast, a curvature larger than cell size was predicted to be below the detection limit of these sensors. Therefore, it is thought that the cytoskeleton, the nucleus, and cell adhesion cooperate to sense the substrate's curvature, but the mechanism is still not fully understood. Chapter 10 discusses the latest findings and approaches based on theoretical calculations and simulations.

In addition to the study of mechanobiology through the control of cell shape, another important aspect was discovering that mesenchymal stem cell differentiation depends on the mechanical properties of the extracellular matrix, as reported by Engler et al. in 2006.⁶⁵  Since the middle of the 20th century, there had been studies suggesting that the substrate's stiffness affects cell survival. However, the authors successfully demonstrated that the elastic modulus of the substrate directed differentiation lineage, including the neurogenesis, myogenesis, and osteogenesis of mesenchymal stem cells (which corresponds to the elastic modulus of actual biological tissues) by using acrylamide gel scaffolds with varying degrees of cross-linking. In addition, nonmuscle myosin II was shown to be involved in the mechanosensing of the cell. Cells sense the mechanical properties of the substrate via intrinsic cellular forces (active touch), indicating that there is an essential cellular force perception in conjunction with the mechanosensing of external forces. The range of cellular stress perception was at the same level as the elastic modulus of biological soft tissues, ranging from 0.1 kPa to 100 kPa, and thus the modulation of various cell and tissue activities by varying substrate elastic moduli was explored. In particular, the acrylamide gel used in this study was familiar to biologists since it is used for SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis). Furthermore, the degree of cross-linking can be easily adjusted, and the material can be treated as a linear elastic material for practical purposes.

The molecular clutch model is currently considered a promising model for describing the mechanism of cellular force perception concerning substrate stiffness. This model is based on the concept that the efficiency of the cellular traction force on the substrate is balanced by a molecular clutch (like that in a car), assuming that the contractile force of actomyosin is the driving force (Figure 1.2).^66,67  Mitchison and Kirschner proposed the original model in 1988 as an example of neurite outgrowth.⁶⁸  In 2008, Chan and Odde established a mathematical model for this phenomenon.⁶⁶  The adaptor protein connecting actin and integrin repeatedly binds and dissociates with the substrate as an elastic body, and the dissociation rate increases with force. The model was validated experimentally and was widely recognised. Later, to resolve the discrepancy between the intrinsic biphasic response of traction force to elastic modulus and the phenotypic diversity depending on the cell type, the existence (concept) of retrograde flow, and the elastic modulus (optimal stiffness) at which the traction force reaches an extreme value was introduced into the model.⁶⁹  Roca-Cusachs et al. demonstrated that this could be explained by the axis of actin–talin–integrin–ECM.⁷⁰ 

On the other hand, the diversity of mechanical properties exists between different tissues and organs and fluctuates along the time axis of biological activities. For example, the mechanical microenvironment surrounding cells dynamically fluctuates during morphogenesis and pathological processes, such as substrate fluidisation during epithelial lumen formation and stiffening of the mesenchyme due to tumorigenesis and fibrosis.^71,72  To mimic such temporal changes in mechanical properties, materials whose elastic moduli change in response to external stimuli have been developed. In particular, hydrogels in which the degree of cross-linking can be controlled enable the design of dynamic substrates that can be softened⁷³  or stiffened⁷⁴  on demand. An interesting concept of mechanical memory is the differentiation behaviour of stem cells depending on the history of the mechanical environment in which the cells are placed was discovered by utilising these properties of the materials.⁷⁵  The design principles and applications of various switchable materials that are responsive to various external stimuli or that can undergo reversible changes are introduced in Chapter 11.

In addition to temporal changes in the elastic modulus of the substrate, cells can sense small gradients in the spatial elastic modulus and appropriately change the direction of cell migration. This phenomenon is known as the durotaxis.⁷⁶  The phenomenon of cell migration towards or against the concentration gradient of molecules in the environment is also known as chemotaxis.⁷⁷  Research has been actively conducted to elucidate the mechanisms of directional sensing and polarity formation. The phenomenon of durotaxis itself is similar to that of chemotaxis; however, it is essential to develop materials that can precisely produce the elastic modulus gradient on a scale smaller than that of a cell to investigate the mechanism of durotaxis in detail. The mechanism by which gradients and absolute values of the elastic modulus of substrates modulate cell migration behaviour is discussed in Chapter 12. This chapter also deals with an emergent phenotype of stem cells, far from the tensional-equilibrated state in normal ones, which has been identified by their repetitive mechanical stimulation from the substrates.

On the other hand, mechanobiology studies on substrate mechanics, which originated from Engler's discovery, have mainly used elastic substrates, but biological tissues exhibit viscoelastic properties. This is because the extracellular matrix composed of collagen is formed by physical cross-linking and is different from elastic hydrogels based on chemical cross-linking, such as polyacrylamide gels. Therefore, studies using viscoelastic materials that mimic living tissues as cell culture substrates have attracted attention. It was found that imparting the viscous properties of the substrate increased cell elongation.⁷⁸  Here, the tuning of the relaxation time of the substrate requires the techniques and insights of material science, and this research has taken a leap forward by the merit of material scientists. Various methodologies have been developed, such as adding dissipative features to the cell culture interface described in Chapter 6 and incorporating dynamic noncovalent bonds into the hydrogel network, as described in Chapter 11. The previously mentioned molecular clutch concept has been extended to understand ECM density and cellular traction forces, and it applies to responses of the viscoelasticity of substrates as previously described.^79,80 

Mechanobiology studies focusing on substrate mechanics have mainly been conducted using two-dimensional (2D) culture systems. In contrast, three-dimensional (3D) culture systems are closer to physiological environments than conventional 2D culture systems and are a promising alternative to bridge the gap between existing cell culture models and biological tissue. Although extensive research has been conducted in tissue engineering, the focus was on properties such as biocompatibility and degradability rather than on the mechanical properties of these 3D scaffold materials. This subject is crucial for addressing and understanding the role of mechanoarchitectonics in vivo from a mechanics perspective. Recent developments in bio-orthogonal chemistry and materials science have led to the successful development of technologies to efficiently encapsulate target cells in hydrogels with tissue-relevant stiffness and high viability. The fact that cells encapsulated in stable hydrogels prepared by chemical cross-linking become mechanically insensitive and undergo a different cell morphology and fate than those in 2D culture systems emphasises the uniqueness of mechanobiology in 3D culture systems. In addition, it has been revealed that cells encapsulated in hydrogels with remodelling and degradation functions similar to those in living tissues can dynamically adapt to changes in the surrounding microenvironment and regulate their functions and fate. Therefore, there is a need to develop materials that can induce dynamic environmental changes in a programmable or adaptive manner in the presence of cells. Such 3D culture systems that enable temporal changes in the mechanical and biochemical environment mimicking in vivo can be regarded as a four-dimensional (4D) culture system and is the new trend in mechanobiology research. Chapter 13 discusses the effects of culture dimension on cultured cells, the design principle of 4D culture systems, and the novel cellular behaviours found in 4D culture systems using hydrogel-based approaches.

Harris et al. observed that wrinkles were formed at the periphery of cell adhesion when cells were cultured on compliant silicone rubber sheets, revealing the presence of cellular traction forces.¹⁰  The detection of these forces applied to molecules and cells, as well as forces and stresses applied to specific sites of cells, is a central issue in mechanobiology, and tools and materials for this purpose have been developed. In detecting molecular dynamics in molecular biology, the approach is to bind to the target molecule and readout its complex formation by fluorescence or luminescence. However, it is necessary to develop various methodologies to detect forces and stresses depending on the application's location, extent, and scale, to resolve cellular mechanoarchitectonics. Many methods have been developed based on the work of Harris et al. to quantify the traction forces applied by a cell to its substrate. Traction force microscopy (TFM) and micropillars are versatile because they can detect force and stress based on finite element analysis from deformation and displacement information using elastic hydrogels and PDMS, as described in Chapter 4. In addition, the development of materials to detect interfacial stress between the cell and the substrate is currently being undertaken, and methodologies that combine the essence of materials science have been proposed, such as membrane-type surface stress sensors (MSS),⁸¹  relying on a membrane-type cantilever, and materials that utilise the piezoelectric emission effect.⁸² 

A fluorescence resonance energy transfer– (FRET-) based fluorescent probe that links a spider-derived peptide sequence with known elasticity to GFP has a significant impact as an approach to detecting the force applied at the molecular level.⁸³  Since this probe is genetically encoded, it can be inserted into the target protein as a chimaera, making it possible to detect the force applied to vinculin, a group of proteins that accumulate in focal adhesions. Because of the convenience of being encoded in a gene, research has been conducted to introduce this module into other proteins, and it has become possible to detect the force applied to α-catenin,⁸⁴  E-cadherin,⁸⁵  etc. Salaita et al. developed a molecular force probe based on DNA nanotechnology, which led to a major revolution, and more details are discussed in Chapter 3.

In addition, it is now possible to detect cell membrane tension using fluorescence probes whose conjugate system expands and contracts in response to tension and measures forces in tissues using perfluorocarbon (PFC) developed by Campàs⁸⁶  and hydrogel-based cell-like microsensors developed by Cappello et al.⁸⁷ 

As previously described, various materials and methodologies for detecting forces and stresses have been developed depending on the difference in the hierarchy of target molecules, cells, and tissues, a trend that will continue in the future.

In concluding this chapter, we would like to highlight the prospects of materials science research contributing to mechanobiology (Figure 1.3). While molecular biology studies unravel the molecular basis of life, mechanobiology considers the effects of “forces” on these phenomena. Mechanobiology complements molecular studies and should actively integrate technological innovations such as super-resolution microscopy (Figures 1.1E and 1.3A)^88,89  and next-generation sequencing. Technologies such as genome editing and iPSCs have brought about significant changes in biology. Chapter 14 describes good examples of using these state-of-the-art technologies to construct and understand pathological models of mechanorelated diseases.

In addition, most of the research in material-based mechanobiology has been conducted analytically in vitro, but future developments are expected to include technologies for controlling and measuring these forces directly in vivo. In such a case, it is essential to design bioinert biomaterials, and the chemical and mechanical design of the interface and bulk needs to be carefully thought out. Moreover, for organoid generation, it is essential to design materials that synchronise the time axis, such as the time evolution of biological phenomena and the mechanical properties of materials. We hypothesise that such “life-adaptive materials” will be the future of material-based mechanobiology (Figure 1.3B). This book focuses on the topic of material-based mechanobiology. Therefore, it mainly discusses materials that explore intracellular forces caused by the contractile force of actomyosin. The topics such as cellular force perception to external shear and stretching stimuli, which are essential and significant themes in mechanobiology, are not covered in detail. However, more active integration of the knowledge of materials science will lead to the development of therapeutic mechanobiological materials and technologies (Figure 1.3C).

Moreover, while information science has been attracting the attention of all fields of science and technology, life science has greatly benefited from bioinformatics advancements in data analysis. The technology of acquiring mechanical information combined with its digital transformation (DX) (Figure 1.3D) and feeding it back to personalised medical technology and preventive medicine and further processing with artificial intelligence (AI) is expected to develop significantly in the next few years.