Mechanotransduction is the process by which cells convert mechanical stimuli into a chemical response [52]. This plays an essential role in the probing of matrix stiffness and is currently subject of extensive research. It is beyond the scope of this review to provide a detailed description of the highly complex signaling cascades involved in mechanotransduction. Instead, we focus on a basic understanding of how cells probe the stiffness of a substrate. For a more detailed explanation we refer the reader to recent reviews on this topic [45, 53-57]. Cells bind to a substrate with transmembrane molecules called integrins, which have an extracellular domain that attaches to the substrate and an intracellular domain that connects to the cytoskeleton (Fig. 2). When cells bind to the ECM, integrins begin to cluster, which leads to the recruitment of structural and signaling proteins to form so-called focal adhesions at the site of integrin clustering. The formation and maturation of focal adhesions requires the application of mechanical forces to these adhesions. Cells can actively generate these forces themselves using actin-myosin complexes which are part of their cytoskeleton. This is where substrate stiffness comes into play. On a hard substrate, cells generate large forces which leads to the formation of mature focal adhesions and a highly organized cytoskeleton with abundant stress fibers [45, 58]. In contrast, a soft substrate cannot provide enough resistance to counterbalance large, cell-generated forces. Therefore, on soft substrates cells do not develop abundant stress fibers and generate smaller forces. In other words, a cell responds to differences in substrate stiffness and adjusts its ‘musculoskeletal system’ (i.e. the cytoskeleton) appropriately.

Fig.(2). Schematic representation of a cell which has attached to a substrate. Cells attach to a substrate with transmembrane molecules called integrins. When cells bind to a substrate, integrins begin to cluster which leads to the formation of focal adhesions (FA). The maturation of focal adhesions requires the application of mechanical forces (F) to these adhesions which can be generated by the cytoskeleton.

Changes in cytoskeletal organization are important, because the cytoskeleton is involved in many signaling pathways that transfer mechanical feedback into chemical responses. Furthermore, the cytoskeleton also determines the shape of a cell, which in turn is intimately connected to cell behavior (reviewed in [56, 59]). This was clearly demonstrated by cell biophysicist Christopher Chen and co-workers who showed that cell morphology can regulate differentiation of stem cells [60]. This group cultured stem cells on adhesive islands with different sizes to force stem cells to adopt a particular cell shape. On small islands, cell spreading was impaired due to space limitations, whereas cells normally spread on larger islands. In culture medium containing both adipogenic and osteogenic differentiation factors, it was found that cells became adipocytes on the small islands but committed to the osteogenic lineage on the large islands [60]. In other words, when these cells received culture medium supporting both osteogenic and adipogenic differentiation, the cell shape determined their fate. Even more intriguing, when cells were cultured on large islands in adipogenic medium, the cells still became osteogenic; vice versa, cells on small islands in osteogenic medium still became adipogenic. Thus, cell shape could drive stem cell differentiation and overrule the instructive messages provided by soluble differentiation factors. The cytoskeleton, in the form of actin-myosin generated tension proved to be essential in the stem cell commitment process.

Since substrate stiffness influences the cytoskeletal organization, it also affects cell morphology. Various studies indicated that stiffer substrates generally promote cell spreading, whereas soft substrates induce a more rounded cell shape (Fig. 3 and refs. [58,61,62]). From the above, it is not surprising that these changes in cell morphology are accompanied by changes in cell behavior, including cell differentiation. Discher and co-workers showed that muscle precursor cells optimally differentiate into myotubes on substrates with a stiffness that mimics that of muscle tissue [63, 64]. In addition, this group demonstrated that substrate stiffness can control the differentiation of stem cells [47]. Naïve mesenchymal stem cells were shown to specify lineage and commit to phenotypes with extreme sensitivity to substrate stiffness. Soft gels that mimic brain tissue are neurogenic, stiffer matrices that mimic muscle tissue are myogenic, and rigid gels prove osteogenic. During the initial week of culturing, reprogramming of these lineages is possible with the addition of soluable induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix stiffness [47].

Fig.(3). Fibroblasts cultured in our lab on ‘soft’ and ‘stiff’ polyacrylamide gels coated with collagen I, four hours after plating. On a soft gel, cells adopted a round morphology whereas these cells spread on stiff gels.

Apart from changes in cell differentiation, the substrate stiffness also affects cell migration. This is important for the ingrowth of cells from host tissues, for example for the vascularization of a scaffold. It was reported that normal rat kidney epithelial cells and fibroblasts migrate faster on softer substrates compared to stiff substrates [49], whereas vascular smooth muscle cells showed maximal migration on intermediate stiff substrates [65]. It was demonstrated that these effects are mediated by changes in focal adhesion formation and myosin-generated forces [49]. Furthermore, when fibroblasts were cultured on a substrate with a soft and a stiff side, they preferred migrating into the direction of the stiff side (a phenomenon called ‘durotaxis’) [66]. Considering tissue formation, it was shown that fibroblasts merge to form tissue-like structures on soft substrates whereas these cells migrated away from one another on stiff substrates [67]. Similar behavior was observed for epithelial cell lines and explants from neonatal rat hearts. The driving force behind this merging of cells on soft substrates is a combination of weak adhesions and myosin II contraction [67].

Substrate stiffness also influences cell growth and viability. For example, osteoblasts showed increased proliferation, motility, and deposition of mineral on hard surfaces compared to soft substrates [68]. Importantly, the responses to substrate stiffness are different for normal and oncogenic cells. Non-transformed fibroblasts showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis on soft substrates [69]. In contrast, transformed cells maintained their growth and apoptosis characteristics regardless of substrate rigidity. This indicates that feedback from substrate stiffness is required for proper regulation of cell growth and survival.

In the polyacrylamide gel system, cells are attached to a substrate as depicted in Fig. (2). This means that the cell only attaches to a substrate on one side, thus experiencing a two-dimensional (2D) situation. Recently, the effect of substrate stiffness on cell behavior was studied in three-dimensional (3D) model systems [70-72], whereby cells are embedded in a gel consisting of ECM proteins. These 3D in vitro model systems provide a better representation of the 3D extracellular environment that cell encounter in vivo [59, 73]. A drawback, however, is that it is difficult to independently control substrate stiffness and ECM biochemistry in these 3D systems. It appeared that matrix stiffness plays an important role in modifying cell behavior also in a 3D environment. For example, smooth muscle cell morphology was much rounder in stiff compared to soft 3D ECM analogs [71]. Interestingly, there can be important differences in cellular responses between 2D and 3D culture models. For example, the migration of human prostate cancer cells in 2D and 3D were shown to exhibit diametrically opposite behaviors [74].

In summary, current data demonstrate that substrate stiffness affects essential cell behavior including differentiation, migration, and growth. The ‘musculoskeletal system’ of the cell, i.e. the cytoskeleton, plays a crucial role in translating feedback from the substrate stiffness into cell behavior.

Only few studies investigated the combined effects of biochemical and biophysical factors on cell behavior. As already mentioned, cell shape and substrate stiffness can supersede biochemical signaling under certain conditions [47, 60]. In addition, soluble induction factors appear to be less selective than matrix stiffness in driving stem cell differentiation, and cannot reprogram stem cells that were precommitted for weeks on a matrix with a certain stiffness [47]. On the other hand, a ‘correct’ substrate stiffness and soluble induction factors can combine to induce a more complete cell differentiation [47]. Considering substrate stiffness and ligand density (i.e. the availability of attachment sites), it was found that smooth muscle cells spread maximally on intermediate ligand densities on stiff substrates [62]. However, on soft substrates these cells were relatively insensitive to ligand density and showed a more rounded morphology [62]. Stimuli induced by specific ECM proteins also appear to be dependent on the stiffness of a substrate. It was found that collagen V, an ECM protein that is highly expressed during tissue development and repair, modifies cell morphology and migration on substrates with a tissue-like stiffness, but not on hard substrates (R.G.M. Breuls, submitted).

When focusing on the mechanical properties, a scaffold should be strong enough to withstand the loads that act on a skeletal tissue at the defect site. Furthermore, the stiffness is important because it affects the strains acting on a cell while being attached to a scaffold [76]. For example, an extremely stiff titanium scaffold will clearly not transfer external loads to cells because it will not deform under physiologic loading conditions. However, the influence of mechanics on cell behavior is not limited to external loading. Even in the absence of external loads, the stiffness of a scaffold is important for the regulation of cell behavior.

Since substrate stiffness affects many different processes, such as cell growth, migration, and differentiation, it is difficult to provide a general guideline for a suitable scaffold stiffness that optimally stimulates tissue regeneration. Nevertheless, to optimally promote stem cell differentiation, current knowledge suggests that the scaffold stiffness should match the in vivo stiffness of the skeletal tissue under consideration. It is important to emphasize that a scaffold should probably exhibit the stiffness of a developing skeletal tissue, which might be lower than the stiffness of a mature tissue. To promote the invasion of the scaffold with cells from surrounding host tissue, a stiffer scaffold might be more favorable [66]. This may set conflicting requirements for the scaffold stiffness with respect to stem cell differentiation and invasion of host cells.

Since substrate stiffness and biochemical stimuli interact to determine cell behavior, both parameters need to be controlled in scaffold design. Natural polymers such as collagen provide ligands for cell binding, however, the mechanical properties of these natural polymers are difficult to control. Synthetic polymers on the other hand generally offer the advantage of better control over the material properties but may require additional treatment to incorporate specific cell binding sites. By determination of the specific amino acid sequences of natural proteins that cells bind to, researchers were able to mimic cell binding sites in custom made peptide sequences that can be incorporated in synthetic scaffolds [77, 78]. Therefore, latest trends in scaffold design have been focusing on incorporating spatially well-defined cell binding sites in synthetic materials [79-82]. Such synthetic mimics of natural ECM materials have already been successfully applied for the regeneration of bone defects in rats [83, 84].

Most research on the use of stem cells in skeletal tissue engineering applications have relied on bone marrow derived stem cells. The use of stem cells extracted from bone marrow may require culturing of the stem cells in vitro in order to enrich and expand the stem cell population. Such procedures are costly and quality insurance in the clinical setting may be difficult to achieve. Cell culturing on conventional hard tissue culturing flasks might introduce another problem, however, since these hard materials can induce an undesired preconditioning of the stem cells as a consequence of an inappropriate stiffness [47]. Therefore, it might be useful to consider expansion of cells on substrates with a stiffness that compares to the stiffness of native tissue. However, cells may not proliferate on soft substrates that mimic native tissue. The problem is that cells either proliferate or synthesize matrix and differentiate at high rates. Stiff substrates seem to enhance proliferation, whereas soft substrates are more dedicated to differentiation.

The introduction of alternative sources for stem cells such as the adipose-derived stem cells may allow for a different approach that solves this problem [85, 86]. Adipose-derived stem cells can be obtained in relative large quantities, which circumvents the need for cell culturing [87-89]. This has led to the formulation of a so-called one-step surgical procedure that avoids in vitro cell culturing [90]. In this procedure, stem cells are harvested form the patient, immediately seeded onto a scaffold, and implanted into the patient, all in a single surgical procedure. In this approach, an appropriate scaffold may circumvent undesired pre-conditioning of stem cells and, ideally, provide biochemical and mechanical stimuli that support the process of stem cell differentiation.

Generally speaking, most synthetic polymers that are currently used in orthopedic practice are stiff materials with elastic moduli in the range of ~3 to 2000 MPa [91]. This suggests that these polymers could be suboptimal to stimulate stem cell differentiation, in particular for softer tissues. The latest trend, however, is that polymers such as PLA are used to create loosely packed fibrous meshes that have a much lower stiffness [92-98]. As a consequence, the cells ‘feel’ a relative low stiffness when they are embedded in these fibrous meshes. This opens up exciting new opportunities with these FDA-approved biodegradable materials. The tissue engineer may fabricate a scaffold with a stiffness that is suitable to promote the desired cell behavior. This might be the stimulation of cell proliferation, differentiation, or even the migration of host cells, depending on the specific requirements in situ. Obviously, a vast amount of research is needed to further investigate the effects of substrate stiffness in combination with other variables such as pore size in the context of these fibrous meshes.