In tissues, cells are embedded within the extracellular microenvironment, a highly hydrated network that comprises three classes of stimuli or cues that stem from the following sources: insoluble hydrated macromolecules (e.g. fibrillar proteins, proteoglycans, or polymer chains), soluble molecules (e.g. growth factors or cytokines), and membrane-associated molecules of neighboring cells [8, 24]. As it is assumed that most interactions between cells and these extracellular effectors are determined by associations between receptors and corresponding ligands [26], we will concentrate here upon specific ligand-receptor interactions and disregard nonspecific effects, such as electrostatic interactions. Ligand-receptor interactions are considered as specific, as they depend on detailed topographical features of interacting structures (“lock-and-key principle”) [26].
Soluble receptor ligands, such as growth factors and cytokines, are thought to diffuse to their target receptors. The transmitted information will arise from the type of signaling molecule as well as its local concentration [19]. The resulting cellular response to that kind of stimulus is currently being investigated in detail; comprehensive reviews dealing with the application of growth factors in tissue engineering can be found in the literature [27–29]. By contrast, the pure biochemical information provided by ligands attached to an extracellular structure, such as the extracellular matrix (ECM), is supplemented by additional degrees of information including the spatial distribution of ligands and the mechanical properties of the structure the ligands are attached to [19]. Spatial variations in adhesiveness, for example, can lead to
2.2 Environmental factors as morphogenetic guides
termed haptotaxis [30–32]. In the following paragraphs, however, we will focus on the impact of mechanical cues on such cell behavior.
2.2.1 Integrins as mechanoreceptors
In the past, great efforts have been made to elucidate how physical forces, applied to either the ECM or the cell surface, induce biochemical alterations inside the cell.
Today, there is much evidence that mechanical signals are transferred into the cell across transmembrane molecules, such as integrins, which couple extracellular anchors to the cytoskeleton [33–35]. Integrins constitute a large family of transmembrane, heterodimeric receptors that bind to specific amino acid sequences, such as the arginine–glycine–aspartic acid (RGD) recognition motif, present in all major ECM proteins [36]. After binding to ECM ligands, integrins cluster together to form dot-like adhesive structures termed focal complexes. Depending on the stiffness of the underlying substrate, focal complexes can disappear or evolve into focal adhesions.
These multi-molecular plaques anchor bundles of actin filaments (stress fibers) and mediate strong adhesion to the substrate. In turn, focal adhesions are considered to be a source for fibrillar adhesions, which are involved in matrix assembly into extracellular fibrils [37, 38]. Studying cell-matrix interactions in a three-dimensional (3-D) context, Cukierman et al. described distinctive “3-D matrix adhesions” that differed from both focal and fibrillar adhesions characterized on two-dimensional (2-D) substrates in structure, localization, and function. They further speculated that classically described in vitro adhesions are exaggerated precursors of those, more biologically relevant “3-D matrix adhesions” [39, 40].
To explain the molecular basis of mechanotransduction, Ingber et al. proposed the cellular tensegrity model [34, 35]. According to this model, living cells are thought to exist in a state of pre-stress, actively generated by myosin-II driven isometric contractions of the actin cytoskeleton. Structural elements that resist compression, notably internal microtubule struts and ECM adhesions, act as a counterbalance. As cell-ECM adhesions and microtubule struts resist cytoskeletal tension in a comple-mentary manner, changes in ECM mechanics or extracellular perturbations generate mechanical forces within the cytoskeletal structure. In reaction to unbalanced forces, cells rearrange cell-matrix adhesions, reorganize their cytoskeleton, and immediately
cytoskeleton, cells will flatten and spread. In the opposite case, cells will retract or become rounded. Biochemical responses are thought to be mediated by conforma-tional changes of regulatory molecules within the adhesion plaque. These molecular events, in turn, trigger signal transduction cascades which ultimately regulate cell proliferation, differentiation, and apoptosis [33, 35].
2.2.2 Mechanical cues regulate cell behavior
Model considerations of cellular mechanosensitivity are also supported by experi-mental data. Using fibronectin-coated beads held in an optical trap, Choquet et al.
demonstrated that cells strengthen their integrin-mediated contacts to the beads in proportion to the force restraining it. According to the authors, this mechanism might allow cells to migrate through the ECM in response to its mechanical properties [41].
In a later study, Lo et al. verified the idea of mechanotaxis by demonstrating that cell movement is guided by the rigidity of the substrate [42]. Similar results were found by Gray et al. They used a micropatterning technique to produce fibronectin-coated surfaces of varying stiffness and observed cell migration towards stiffer regions of the substrate (Figure 2.1) [43].
Figure 2.1: NIH/3T3 fibroblasts cultured on fibronectin-coated poly(dimethylsiloxane) (PDMS) substrates. The squares are stiff, whereas the regions surrounding the squares are compliant. Accumulation of cells on stiffer regions was found to be due to migration, not proliferation, of cells in response to the mechanical patterning (mechanotaxis). Scale bars represent 100µm. Reprinted with permission from Gray et al. [43]. c2003 John Wiley & Sons, Inc.
In addition to migration, a variety of other cell functions, such as cell spreading, growth, and differentiation, are also modulated by the substrate mechanics. Pelham et