An Introduction to Mechanobiology S. D. Dubal and Y. L. Vyas Department of Anatomy, College of Veterinary Sci.& A.H.

, Anand Agricultural University, Anand - 388 001 (Guj) India

If someone asks why we are? It is impossible to give the answer. However, if it is asked what and how we are, there are fair chances of the answer. It may happen that the answer may be of religious philosophical, socio-economical or what is now a day said scientific. Yes, where the religious philosophy ends, the science comes to settle the questions. No human being can have his/her past more than 150 years. It is also difficult to remember the whole past. The ignorance is dangerous but it is the force behind all creativities and innovations to open the gate of knowledge. When we think about how much time lapsed since the appearance of first living organism, our past time span is nothing. At least it is physically plausible to understand the life (the living organism). There are several types of living organisms and lot of variations in the forms and (hence) habitats exit. Nevertheless all the organisms are constructed on same materials and principles. The answer of question ' what are the organisms' is thus simple. The organisms are nothing but chemicals. The chemicals are such that they emerge through hierarchical self-assembly of nanoscale components. This assembly is highly unstable and is always in surge of stability. The stability is achieved under the action of various forces that are mechanical or converted into mechanical forces. Without mechanical forces the life is not possible. The molecules that are assembled were not synthesized in one day. There had been synthesis of different molecules. Amongst all molecules synthesized, a few were had the ability to self-assemble and, which could remodel to form flexible structures under the influence of highly flexible clay minerals (and hence they can catalyze chemical reactions with greater efficiency, including reactions that produced the first organic polymers). This took millions of years to have such molecules. The assembly which could remain persisted gave the birth of the living organisms. This was the beginning of survival of fittest. The surge for stability is still continuing. Without a cellular structure as the basis of life, there is no basis for cell growth and division and therefore evolution and diversification would likely have been impossible. Living cells are the ultimate intelligent materials. They are mechanically strong and resilient; exhibit integrated multifunctional chemical/mechanical/electrical/optical and information-processing capabilities; grow, move and self-heal; learn, adapt, and self-organize (Inber, 2000). As a first priority, they required mechanisms that would protect their delicate scaffold from potentially damaging stimuli. However, to interact with their changing environment (e.g., during feeding, escaping, or mating), they needed mechanosensitivity. On the basis of mechanical properties of the surroundings, the cell performs the physical and chemical activities so that it can remodel. The continuous process of

remodeling leads to survival of the cell (organism).This is the evolution. During the evolution, the cell developed its own scaffold that bridged the extracellular materials (matrix - ECM) and central body that has the ability to control the synthesis of desired molecules as per the mechanical stimuli. Thus the whole assembly is dependent upon the recognition of the external mechanical stimuli. In other word, the mechanical stimuli are necessary for the livingness of the cell. The following pages concern the present day multicellular organisms. Cytoskeleton: The simple understanding about the structure of a cell is that it is composed of cell membrane and nucleus (bound by nuclear membrane). Between them lies the cytoplasm, which is in the form of interchangeable sol- gel phase and the organelles and several inclusions form the cytoplasm. Due to the use of point-loading, cells have a structural framework to transmit forces from one place to another inside their cytoplasm. This structure, known as the cytoskeleton (CSK), is a highly interconnected three dimensional network composed of three major biopolymer systems, microfilaments, microtubules, and intermediate filaments along with their associated proteins. Microfilaments contain polymerized actin. Actin filaments are continuously under tension that is continuously transmitted throughout the entire interconnected actin lattice and thus, the whole cell. Microtubules are hollow tubular polymers composed of different tubulin monomer isotypes and resist compression. Growing microtubule buckles under compression. The buckling occurs when the microtubule elongates and pushes against other components of the cells skeleton. Intermediate filaments like actin filaments, they function in the maintenance of cell-shape by bearing tension. Intermediate filaments organize the internal 3D structure of the cell, anchoring organelles and serving as structural components (e.g., vimentin, desmin, cytokeratin). N atural Design Principles: Cytoskeleton is based on the following Natural Design Principles (Ingber, 2000): 1. Minimize energy expenditure. 2. Obey spatial constraints. 3. Develop emergent properties through architecture. 4. Establish a mechanical equilibrium. 5. Use discrete networks. 6. Maximize tensile materials. 7. Stabilize through triangulation or prestress. 8. Use structural hierarchies. 9. Develop self-renewing functional webs through emergence of autocatalytic sets. 10. Enhance functional efficiency through solid-state biochemistry. Tensegrity: All of the basic design principles listed above are embodied in one architectural system that is known as tensegrity. Tensegrity is a prestressed structure, having architectural system, in which, structures stabilize themselves by balancing the counteracting forces of compression and tension. It gives shape and strength to both natural and artificial forms ((Ingber, 2008). The necessary and sufficient conditions, for

a Tensegrity structure, are: (i) it distributes stresses to establish a force balance and (ii) stabilizes itself against shape distortion. An internal molecular framework (Tensegrity Structure) is composed of actin microfilaments, microtubules and intermediate filaments that give shape to the cell. The Tensegrity Structure of the cytoskeleton can be changed by altering the balance of physical forces transmitted across the cell surface. This finding is important because many of the enzymes and other substances that control protein synthesis, energy conversion and growth in the cell are physically immobilized on the cytoskeleton. For this reason, changing cytoskeletal geometry and mechanics could affect biochemical reactions and even alter the genes that are activated and thus the proteins that are made. Extra-cellular m atrix (ECM ): The interaction of cells with the surrounding extracellular matrix (ECM) is essential in many physiological and pathological processes, such as embryonic development, maintenance of tissue homoeostasis, repair, fibrosis, tumour invasion and metastasis. Besides providing tissues with structural integrity, ECM proteins form the three-dimensional scaffold needed for cell adhesion and migration. The interaction of a three-dimensional scaffold with specific receptors at the cell surface, the correct composition and pliability of the ECM are prerequisites for the proper molecular structure and functioning of connective tissues. This also requires effective bi-directional signal transmission between ECM molecules, cell surface receptors, e.g. integrins, and the cytoplasm. Presence or absence of physical stimuli have been shown to trigger adaptive responses in vitro as well as in vivo. Integration of these complex signaling networks leads to tight regulation of the cellular metabolism and controls a number of functional properties of various cell types Mechanical forces stimulate cell differentiation and control/maintain tissue function via membrane associated mechanoreceptor mechanisms, activation of second messengers and downstream gene regulation in various mammalian systems like musculoskeletal and cardiovascular tissues. Cell adhesion molecules like integrins, membrane associated receptors and strain-stress sensitive (mechanically-gated) ion channels transmit mechanical forces to intracellular structures like the actin cytoskeleton and second messengers like calcium flux (Ingber, 2003). Tissues with cells are living materials, which have the ability to sense their physical environment and react with adaptive responses (Vogel and Sheetz, 2006). Therefore, the cell has some mechanical properties to sustain the viability. There are the older sciences now known as Biomechanics and Biorheology (the study of the flow properties of biological fluids). Mechanobiology is a rather young research field which is closely related to biomechanics and biorheology. The link between these two research fields is a process called M echanotransduction. Biomechanics investigates the whole body or parts of the body and their functions from a mechanical viewpoint or in other words, biomechanics seeks to understand the mechanics of living systems (Fung 2004). Recent achievements of applied biomechanics have helped to solve problems not only in orthopedics but in various other areas of regenerative medicine. Biomechanics also deals with the measurement of the material properties of different tissues (the load, determine the stresses, interstitial fluid flow and strains within the tissue), which is an important input for mechanobiological studies.

On the other hand, Mechanobiology wants to predict growth and differentiation of tissue and cells in quantitative terms, based on a given force exerted on a given tissue matrix populated by cells (van der Meulen and Huiskes 2002). Thus, Mechanobiology is mainly concerned with the study of the influence of mechanical forces on cells and tissues and their clinical or therapeutical applications. Naturally, mechanobiological research is based on interdisciplinary approaches. There are numerous examples that show the effect of force on various biological processes. Mechanical forces (for example, muscle contraction) are essential for healthy embryonic skeletal development. In the absence of muscle contraction in mouse, there is loss of shoulder and elbow joints (Kahn et al., 2009); there is cartilaginous fusion of the femur and tibia in stifle joint of chick (Roddy et al., 2011). Further more, treatment with agents (e.g., Y27632, cytochalasin D; 2,3-butanedione 2-monoxime (BDM) and ML9) that disrupt or suppress cytoskeletal tension generation by various mechanisms, inhibit lung morphogenesis in mouse (Ingber, 2006). Mechanical strain on bone marrow stromal cells induces proliferation and differentiation into osteoblast-like cells. Static control and short time strained cells display no mineralization at all. After long time straining, cells showed significant mineralization (Griensven et al., 2005) The structure and function of tendon and ligament are sensitive to alterations in mechanical loading. Immobilization results in decreased tendon size and strength. This strength slightly return after tendons are exercised (Lujan, 2010). Mechanical loading also affects the postnatal development of the supraspinatus tendon-to-bone attachment (enthesis) (Thomopoulos et al., 2010). The collagen fiber distribution is less organized in unloaded tendon in comparison to the loaded tendon. Ultimate stress is significantly lower in the unloaded tendon in comparison to the loaded tendon. Bone adapts during skeletal growth and development by continuously adjusting skeletal mass and architecture to changing mechanical environments. The changes may be summarized into three fundamental rules that govern bone adaptation (Turner, 1998): 1. It is driven by dynamic, rather than static, loading. 2. Only a short duration of mechanical loading is necessary to initiate an adaptive response. 3. Bone cells are less responsive to routine mechanical loading signals. Living cells that continually remodel bone are able to sense changes in mechanical stresses in their local environment and that they respond by depositing new extra-cellular matrix ECM where it is needed and removing it from where it is not (W olff s Law). Mechanical force (load) affects the organization of the network of trabeculae bone (Chen and Ingber, 1999). Trabeculae bones are arranged along the line of action of force and provide maximum strength. It is one of the most beautiful examples of the importance of cellular mechanotransduction for regulation of tissue morphogenesis. Mechanical loading is a critical factor in proper differentiation of stem cells into cartilage cells (chondrocytes) that synthesis load-bearing tissue. Dynamic loads are more advantageous than static loads. Similarly, blood vessels, when exposed to pulsating loads,

the smooth muscle cells align uniformly at a 65 angle to the applied force. Cyclic stretching induces the production of collagen and proteoglycans (Lujan, 2010 ) In immobilized stifle joints, expression of the COL2A1 and TNC mRNA genes are adversely affected. There is absence of the inter-articular ligaments, the chondrogenous layers, the joint capsule, patella region and menisci (Roddy et al., 2011). Further more, cyclic strain induces actin reorganization, nuclear translocation of MAL/MRTF-A and tenascin-C expression in fibroblasts (Chiquet et al., 2009). The mechanosensitive responces of stem cell for differentiation depend on the biomechanical properties of ECM (Engler et al., 2006). Mesenchymal Stem Cells (MSCs) are platted onto collagen-coated gels of varying stiffness. On soft, collagencoated gels that mimic brain elasticity (0.1-1 kPa), the vast majority of MSCs adhere, spread, and exhibit an increasingly branched, filopodia-rich morphology. MSCs on 10 fold stiffer matrices that mimic striated muscle elasticity (8-17 kPa) lead to spindle shaped cells similar in shape to myoblasts. Stiffer matrices (25-40kPa) that mimic the crosslinked collagen of osteoids yield polygonal MSCs similar in morphology to osteoblasts. M echanotransduction: The mechanical stimuli are converted into a physiological phenomenon by the following processes: 1. Mechanical coupling: The transformation of the applied force into a force which is detectable by the cells or the induction of a physical phenomenon. 2. Mechanotransduction: the process by which cells sense and respond to mechanical signals and is mediated by extracelluar matrix (ECM), transmembrane integrin receptors, cytoskeletal structures (CSK) and associated signaling molecules. 3. Signal transduction: The conversion of the mechanical signal into intracellular physiological signals. 4. Cellular response: Regulation of a gene, release of autocrine or paracrine factors, expression specific receptors. M echanotransduction and Integrins: Cells are linked together by extracellular matrix (ECM) scaffolds. Cell-matrix adhesion (the interaction between cells and the components of ECM) contacts act as sites of mechanosensation. Cells adhere to the ECMs (mainly composed of laminin, collagen, fibronectin, elastin, glycoproteins and proteoglycans) via binding of specific cell surface receptors, known as integrins. Integrins molecules are present on the surface of the cell membrane and form a multi-molecular bridge between the ECMs and internal cytoskeleton of the cell. Many of the actin filaments closely associate with myosin filaments, which slide along each other, shorten and thereby, generate mechanical tension that is distributed to all elements of the cell, as well as to the external ECM, via their integrin contact points. Moreover, forces transmitted over these integrins are converted into changes in the form of intracellular biochemistry and gene expression. The cellmatrix adhesions regulate the shape and activity of the cell (cell migration, growth, and differentiation); determine 3-D organization of tissues and organs during embryonic development and play a role in cancer formation (Geiger et al., 2001 and 2009; Ingber, 2008).

Integrins: It is the most important receptor that attaches cells to their extracellular microenvironment. The interaction is likely: ECM-Integrins-Linkers (adaptors)cytoskeleton-Nuclear membrane- Gene expressions.

Structure of the Integrin: E xtracellular domain: is composed of dimer of alpha (17 types) and beta (8 types) subunits linked non-covalently. The alpha subunit has 7-bladed b propeller and I-domain; beta subunit has I-domain only. There is RGD binding site on I-domain of beta unit. Ca2+ are required to maintain structure. Intracellular domain: It binds to linkers like Vinculin and Talin--linkers to actin. Also binds Plectin which links to keratin intermediate filaments. ECM-integrin interaction activates FAK. Integrins coordinate regulation of exogenous tension (matrix rigidity) and endogenous tension (contractility). The results are: A: Cells and matrix mutually interact to regulate tension. B: Tension in the cell microenvironment is distributed by integrin receptors that signal bi directionally between extracellular and intracellular compartments. Tension levels may alter outside-in and inside-out integrin signaling. Integrin engagement with extracellular matrix (ECM) regulates endogenous cellular tension by triggering actin cytoskeletal organization and acto-myosin contractility. Endogenous tension levels can indirectly or directly control exogenous tension (matrix rigidity). M echanotransduction Response: The network including integrin and the cytoskeleton is activated by mechanical stress and causes binding of cytoskeletal elements such as talin, paxcillin (PAX), vinculin (VIN), or focal adhesion kinase (FAK), eventually leading to transcription factor activation. The GTPase Rho causes realignment of actin stress fibers during FSS and is associated with induction of gene expression . Diseases of M echanotransduction: Not all but most of diseases of heart, GIT, kidneys, eyes, organs of nervous system, skin, lung, diabetes, pediatric and cancers are rooted into the mechanotransduction. The obvious causes lie at the level of ECM, Integrins, Lnkers CSK and Nucleus (including nuclear membrane). New drugs are targeted at these entry levels to cure the tedious and fatal diseases. Applications of Mechanobiology: There are several applications for the treatment of diseases. The following are a few examples: 1. Fracture fixation now emphasizes micromotion to accelerate new bone formation. There is improved callus formation when micromotion occurs at fracture (Lujan, 2010). 2. Future techniques to reduce bone resorption. i. Distraction osteogenesis (limb lengthening) ii. Osteoporosis: Mechanical loading of bones is taking a central role in treatment and prevention of this disease. 3. Cross-fiber massage performed on ligament after injury, improves fiber orientation and strength (Lujan, 2010).

4. Skin wound healing: Skin wound healing takes a long time when there is no fluid pouring on the wound. The laminar fluid flow enhances the wound healing, while the turbulence fluid flow takes longer time than the laminar fluid flow (but the time is shorter than the time taken by the no fluid flow therapy). 5. Most of the physiotherapies are based on the proper knowledge of mechanobiology. 6. Diagnosis of Cancer cell Metastasis: Simulation studies indicate that the most important factor in the metastasis of cancer cells is the cell-matrix adhesions. An altered ECM enhances the metastasis of cancer cells. Reestablishment of biomechanical properties of ECM prevents the metastasis (Chaplain et al., 2011). 7. The knowledge of mechanobiology is assisting efforts in tissue engineering. Bioreactors are being used to apply loading regimes during tissue culture to engineer functional constructs that can withstand large and recurring forces. In future there will be developing of multiscale models for the cancerous cell metastasis. The molecules those mediate mechanotransduction including ECM molecules may represent future targets for therapeutic innervations (Makale, 2007). 8. Therapeutic strategies of left ventricle of heart should include either a complete and continuous reduction of load or normalization of left ventricular mass by interventions aimed at specific targets involved in mechanotransduction (Tavi et al., 2001).

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