**3. The Role of Matrix Stiffness in Triggering MSCs' Osteogenic Differentiation**

Matrix stiffness regulates the MSCs' differentiation into mature specific cells by activating transcription factors that upregulate genes responsible for the initiation and progression of particular cell-linage differentiation. The singling pathways involved in MSCs' osteogenic differentiation are illustrated in (Figure 3). Rigid matrices led to increased MSCs spreading and improved actomyosin contractility, promoting osteogenic differentiation. This enhanced potential was accompanied by increased Runx2, β-catenin, and Smad, implying the significant impact of mechanosensing the matrix stiffness and its role in determining the cell fate [41,50]. The relation between Runx2 expression, owing to mechanosensation with actomyosin contractility, was confirmed by inhibiting myosin, which caused a decrease in Runx2 expression [118]. However, the effect of matrix stiffness on MSCs' differentiation disappeared at the monolayer state [49].

The hippo pathway is one of the signaling pathways involved in MSCs' differentiation and is regulated by intra- and extracellular signals [119]. The downstream effectors of the hippo signaling pathway are yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) [120]. YAP and TAZ transduce signals necessary for determining MSCs' fate. The control of the Hippo pathway is through phosphorylation and nuclear translocation of YAP/TAZ [121]. Additionally, matrix stiffness can control the localization and activity of YAP/TAZ [122,123], which is identified through the structural and functional regulation of the cell cytoskeleton to adjust cellular tension [124]. The stresses sensed by MSCs are transmitted to the nucleus and lead to an increase in the nuclear membrane tension, causing expansion of the nuclear pores, which promote sudden nuclear inflow of YAP [125]. In MSCs cultured on a rigid matrix (40 kPa) undergoing osteogenic differentiation, YAP/TAZ has been localized in the nucleus. In comparison, MSCs cultured on a soft matrix (0.7 kPa), YAP/TAZ persisted in the cytoplasm, directing MSCs to undergo adipogenic differentiation [122]. Moreover, YAP knocking-down

resulted in inhibition of osteogenesis and enhancement of adipogenesis [126]. During MSCs' osteogenic differentiation, TAZ functions as a co-activator of Runx2 to stimulate osteogenesis and inhibits PPAR-γ, which reduces adipogenic differentiation [127]. These findings highlight the significant role of YAP/TAZ as a potent regulator of stiffness-induced osteogenic differentiation. *Polymers* **2021**, *13*, x FOR PEER REVIEW 10 of 34

**Figure 3.** signaling pathways involved in stiffness induced MSCs' osteogenic differentiation. **Figure 3.** Signaling pathways involved in stiffness induced MSCs' osteogenic differentiation.

The hippo pathway is one of the signaling pathways involved in MSCs' differentiation and is regulated by intra- and extracellular signals [119]. The downstream effectors of the hippo signaling pathway are yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) [120]. YAP and TAZ transduce signals necessary for determining MSCs' fate. The control of the Hippo pathway is through phosphorylation and nuclear translocation of YAP/TAZ [121]. Additionally, matrix stiffness can control the localization and activity of YAP/TAZ [122,123], which is identified through the structural and functional regulation of the cell cytoskeleton to adjust cellular tension [124]. The stresses sensed by MSCs are transmitted to the nucleus and lead to an increase in the nuclear membrane tension, causing expansion of the nuclear pores, which promote sudden nuclear inflow of YAP [125]. In MSCs cultured on a rigid matrix (40 kPa) undergoing osteogenic differentiation, YAP/TAZ has been localized in the nucleus. In MSCs fate is also directed through actomyosin contractility and activated Rho/Rho kinase (ROCK) signaling [88], along with mechanotransduction mediated by focal adhesion and integrin [128]. In response to increased stiffness, activated Rho stimulates actomyosin stress fiber assembly [129], which causes an increase in cell contractility and activation of ERK, promoting osteogenic differentiation [130]. Furthermore, Rho combined with the actin cytoskeleton is essential to maintain nuclear YAP/TAZ in MSCs [122]. Activation of FAK via ROCK signaling led to upregulation of osteogenic marker Runx2, ALP, and matrix mineralization denoting osteogenesis of human adipose stem/progenitor cells [131]. In addition, the inhibition of FAK and ROCK signaling caused an upregulation of adipogenic markers. Furthermore, matrix stiffness modulates MSCs' osteogenic differentiation through the Ras pathway, which is accompanied by an increase in the phosphorylation levels of Smad1/5/8, AKT and ERK [49]. Ras (RasN17) inhibition resulted in a significant decrease of Smad1/5/8, AKT, and ERK activity, as well as osteogenic markers' expression [49].

comparison, MSCs cultured on a soft matrix (0.7 kPa), YAP/TAZ persisted in the cyto-

[126]. During MSCs' osteogenic differentiation, TAZ functions as a co-activator of Runx2 to stimulate osteogenesis and inhibits PPAR-γ, which reduces adipogenic differentiation [127]. These findings highlight the significant role of YAP/TAZ as a potent regulator of

MSCs fate is also directed through actomyosin contractility and activated Rho/Rho kinase (ROCK) signaling [88], along with mechanotransduction mediated by focal adhesion and integrin [128]. In response to increased stiffness, activated Rho stimulates ac-

stiffness-induced osteogenic differentiation.

Cells on stiff matrices develop high cytoskeletal tension, which is evidenced by enhanced actin stress fibers and large spread area. Below a compressive modulus of 25 kPa, regardless of the adhesive ligand presented, there is not enough cytoskeletal tension to promote osteogenic lineage differentiation [88]. Based on these results, it has been postulated that, unless a cell develops cytoskeletal tension exceeding a certain threshold stiffness (substrates with moduli of ≥ 25 kPa), osteogenic differentiation will not occur and the cell would need the presence of an osteogenic ligand for Runx2 expression for further differentiation to take place. On the other hand, MyoD1 (a marker for myoblasts) expression demonstrated less ECM dependence compared with Runx2, as it was markedly expressed in cells cultivated on substrates with stiffnesses higher than 9 kPa, regardless of the protein coating [132]. Additionally, on soft poly(acrylamide-co-acrylic acid) substrates (E = 15.4 kPa) that mimic muscle elasticity when grafted with arginine–glycine– aspartate (RGD) peptide sequence, MSCs were directed to a spindle-shaped morphology similar to C2C12 myoblasts, while stiffer matrices (E = 47.5 kPa) that mimic osteoid tissue's crosslinked collagen yield the cells in polygonal morphology, similar to MC3T3-E1 pre-osteoblasts [118].

Inflammation can further counteract the inductive effect of matrix stiffness on osteogenic differentiation. Periodontal ligament stem cells (PDLSCs) cultured with the inflammatory cytokine interleukin (IL)-1β on gelatin/methacrylate hydrogels with different matrix stiffness showed a marked reduction in matrix stiffness-dependent osteogenic differentiation and expression of osteocalcin, as well as Runx2. This was through the activation of p38 signaling pathways, which were activated by IL-1β [133]. Further, macrophages encapsulated in gelatin/methacrylate hydrogels with high stiffness showed a high tendency to polarize toward the pro-inflammatory M1 phenotype, which was associated with a negative impact on the osteogenic differentiation of bone-marrow mesenchymal stem cells (BMMSCs) [134].
