*2.1. Focal Adhesion and Integrins*

Focal adhesions are complexes of highly specialized proteins and macromolecules that can attach cells to the ECM, allowing them to sense and respond to mechanical stimuli [63]. Focal adhesion is composed of the transmembrane protein integrin, which has intracellular and extracellular domains (Figure 1). Integrin's intracellular domain is linked to the actin cytoskeleton via cytoplasmic adapter proteins, which include the actin-binding proteins α-actinin, vinculin, and talin [64]. The integrin extracellular domain binds to ECM components, such as collagen, laminin, and fibronectin, via its extracellular domain, thereby establishing a mechanical connection between ECM and intracellular cytoskeleton components [64–66]. Fifty different proteins have been associated with focal adhesion [67], including intracellular proteins as focal adhesion kinase (FAK) and p130Cas [68,69].

Focal adhesions are complexes of highly specialized proteins and macromolecules that can attach cells to the ECM, allowing them to sense and respond to mechanical stimuli [63]. Focal adhesion is composed of the transmembrane protein integrin, which has intracellular and extracellular domains (Figure 1). Integrin's intracellular domain is linked to the actin cytoskeleton via cytoplasmic adapter proteins, which include the actin-binding proteins α-actinin, vinculin, and talin [64]. The integrin extracellular domain binds to ECM components, such as collagen, laminin, and fibronectin, via its extracellular domain, thereby establishing a mechanical connection between ECM and intracellular cytoskeleton components [64–66]. Fifty different proteins have been associated with focal adhesion [67], including intracellular proteins as focal adhesion kinase (FAK) and

**Figure 1.** Elements of focal adhesion. **Figure 1.** Elements of focal adhesion.

*2.1. Focal Adhesion and Integrins* 

p130Cas [68,69].

Integrins are alpha and beta subunits heterodimers existing in different combinations [64]. There are 18α and 8β subunits, which account for 24 different integrin heterodimers in mammals specific to an exact set of ECM ligands [70,71]. Through their intracellular and extracellular domains, integrins are capable of joining intracellular cytoskeleton with the external environment, thereby creating mechanical integration between ECM and intracellular cytoskeleton [72]. They can transmit cellular signals to the ECM, and reciprocally can convey signals from the ECM intracellularly [73], triggering an intracellular signaling pathway, resulting in alteration of cellular migration, proliferation, and differentiation [74].

It is noteworthy that MSCs' surface integrins' expression can influence MSCs' lineage commitment [75]. Further, matrix stiffness can influence integrin expression on MSCs, which can dictate and direct stem/progenitor cell fate [76]. Undifferentiated MSCs were found to mostly express α1, α3, αV, β1, and β2 integrins, while α2, α4, α5, α6, β3, β4, and

β5 were expressed to a lesser extent [77]. MSCs' osteogenic differentiation was reported to be associated with upregulation of integrin α5 expression on MSCs' surface in response to ECM stimuli [77–80]. Integrin α5 upregulation promotes osteogenesis through activation of FAK via the ERK1/2-MAPKs and PI3K signaling [79]. MSCs' expression of integrin subunits α2 [62,76], α1, αV, and β3 was also upregulated with increased ECM stiffness, favoring osteogenic differentiation [62], while α5 and β1 expression was upregulated in the matrix with lower stiffness [76]. Additionally, activation of MSCs expression of α5β1 and αVβ3 integrin complexes in response to ECM morphology was associated with enhanced osteogenic differentiation [81]. On the other hand, osteogenic differentiation was associated with reduced expression of α1, α3, α4, β3, and β4 integrin subunits [77], while MSCs' adipogenic differentiation was associated with the upregulation of α6 and reduction of α2, α4, α3, β3, and β4 integrin subunits expression [77]. Increased integrin α5 expression can also inhibit both adipogenic and chondrogenic differentiation, while promoting MSCs osteogenic differentiation [82].

The binding of integrin to ECM components triggers the intracytoplasmic assembly of focal adhesion proteins, including talin, FAK, p130Cas, and vinculin, first forming focal complexes, which then grow, giving rise to focal adhesions, linking actin fibers to ECM components [83]. Formation of focal adhesion, with the associated triggering of intracellular signaling pathways, is essential for MSCs migration, proliferation and differentiation [84–86].

MSCs lineage commitment and osteogenic differentiation in response to ECM mechanical cues, including matrix stiffness, involve upregulation of focal adhesion formation. Increasing matrix stiffness can promote number [84] and area of focal adhesions [87]. In turn, upregulation of focal adhesion number and size [60,88,89] has been linked to increased osteogenic differentiation of MSCs. Additionally, tightly packed focal adhesion can stimulate osteogenic differentiation [90].
