*2.2. Cytoskeleton Elements*

Cytoskeletal-related proteins are responsible for the ability of stem/progenitor cells to respond to mechanical cues, including stiffness of the ECM [85]. In addition to their role in providing a cellular structural framework, repolarization of cytoskeleton elements in response to mechanical stimuli, transmits the signals from the ECM to the nucleus, resulting eventually in altered gene expression [64,91,92]. Structural elements of the cellular cytoskeleton include microfilaments, intermediate filaments, and microtubules [64].

The actin cytoskeleton is responsible for the maintenance of cell shape, motility, and contractility. They also act as mechanical sensors for the extracellular environment [93]. It is formed of F-actin, which is a helical polymer of G-actin coupled with actin-binding and actin-bundling proteins, such as α-actinin, vinculin, and talin [94] (Figure 2). Actin cytoskeleton forms a web in association with cellular junctions and forms a core of microvilli, filopodia, and lamellipodia [64]. Actin perinuclear cap is a dome-like structure formed of contractile actin filament and phosphorylated myosin, covering the top of the nucleus and connected to the nucleus through linkers of nucleoskeleton and cytoskeleton (LINC) protein complexes [95,96]. This actin perinuclear cap provides a mechanism through which mechanical signals, transduced through focal adhesion, can reach the nucleus to induce cellular responses [95,97]. Ultimately, the actin perinuclear cap is responsible for conveying signals regarding matrix stiffness to the nucleus [97].

The binding of MSCs to a stiff substrate induces actin polymerization, as evident by an increased ratio of F-actin to G-actin, forming actin stress fibers, which trigger intracellularsignaling pathways [67]. Stress fibers are actomyosin complex composed of F-actin and myosin-2 stabilized by crosslinking proteins [98] (Figure 2). The process of actin polymerization is regulated by the FAK signaling pathway [99]. Actin polymerization and stress fibers formation are essential for establishing cell to ECM interaction [100]. Polymerization of actin dictates lineage commitment of MSCs, as actin depolymerization was noticed during adipogenic differentiation [101]. On the contrary, actin polymerization combined with

an increased ratio of F-actin to G-actin upregulated osteogenic differentiation [99,102–105]. On the other hand, disruption of actin polymerization can reduce osteogenic differentiation [103]. Increased osteogenic differentiation on stiffer substrates was also associated with increased expression of F-actin [78], in addition to actin-binding protein (vinculin) [106]. *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 34

**Figure 2.** Focal adhesion formation and stress fibers assembly in mechano-active cells. **Figure 2.** Focal adhesion formation and stress fibers assembly in mechano-active cells.

*2.3. Mechanosensitive Ion Channels*  Mechanosensitive ion channels are a further mechanism implicated in MSCs' mechanotransduction on stiff matrices. These ion channels are sensitive to substrate stiffness. Upon mechanical stimulation, they allow the intracellular influx of ions and can form complexes with stress fibers, eliciting intracellular signaling pathways [108]. Me-Actin filaments can also interact with other components of the cellular cytoskeleton as intermediate filaments [64]. Intermediate filaments have a diameter of about 10 nm and have a role in maintaining cell shape and cellular junctions [64]. F-actin promotes intermediate filaments and vinculin assembly and disassembly, which are required for the process of osteogenesis through the transient receptor potential melastatin 7–osterix axis [107].

chanical stimulation affects cell differentiation through a change in calcium influx through activated channels [109]. The change in the calcium influx results in the activa-

Different age-dependent changes in MSCs were reported, such as decreased proliferation ability [111] and osteogenic differentiation potential [112–114]. Moreover, age-associated bone loss was linked to the reduced osteogenic potential of MSCs [115]. Aged multipotent progenitor cells lose their sensitivity to alterations in polyacrylamide substrates, while younger multipotent progenitor cells showed a lineage-dependent re-

tion of the MAPK signaling pathway [110].
