**1. Introduction**

Stem/progenitor cells are characterized by their outstanding differentiation potential into multiple types of specialized cell lineages, relying on their pluri- or multipotency, while maintaining their self-replicating ability [1–10]. Among the different stem/progenitor cell types, mesenchymal stem/progenitor cells (MSCs) have been widely used in tissue engineering, cell transplantation, and immunotherapy [11–14]. MSCs were initially isolated from the bone marrow, but can be currently isolated from almost every tissue in the body [15]. MSCs niches are located in different sites, including umbilical cord blood [16], menses blood [17], dental tissues [18], synovial fluid [19], adipose tissues [14], and dental tissues [4]. MSCs reside adjacent to vessel walls, near perivascular regions, on the endosteal surfaces of trabecular bone, and within the interfibrillar spaces [11].

Proliferation and differentiation of MSCs can be triggered by certain growth factors and chemicals, inducing specific genetic events, affecting the release of transcriptional factors, which regulate the differentiation of MSCs into specific lineages [14,17]. Additionally,

**Citation:** El-Rashidy, A.A.; El Moshy, S.; Radwan, I.A.; Rady, D.; Abbass, M.M.S.; Dörfer, C.E.; Fawzy El-Sayed, K.M. Effect of Polymeric Matrix Stiffness on Osteogenic Differentiation of Mesenchymal Stem/Progenitor Cells: Concise Review. *Polymers* **2021**, *13*, 2950. https://doi.org/10.3390/ polym13172950

Academic Editor: José Miguel Ferri

Received: 16 July 2021 Accepted: 5 August 2021 Published: 31 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

biomaterial scaffolds can create a microenvironment that provides MSCs with appropriate conditions for directed differentiation [14]. MSCs further can secrete various immunomodulatory molecules, including cytokines, chemokines, and growth factors, which provide the self-regulated regenerative microenvironment for different injured tissues or organs [13,17].

Regeneration and healing of bone injuries, particularly in large bony defects, is a complicated process [11]. Based on the multipotency of MSCs, they can give rise to either osteoblasts, chondrocytes, myoblasts, or adipocytes in response to key transcriptional regulators that control the primary commitment and most of the follow-up differentiation [20]. MSCs further interact with the components of their local microenvironment (niche) of the extracellular matrix (ECM) [21].

ECM was earlier believed to be an inert matrix that only provides physical support to cells; later, the important role of ECM in various cellular processes was introduced [22]. The MSCs niche provides extrinsic signals including growth factors, ECM, and those released due to contact with other cells. Through these signals, the MSCs' niche could regulate the stem/progenitor cells' fate [23,24]. In this context, interactions of MSCs with their niche are reciprocal; thus, MSCs are capable of remodeling the niche in response to signals received from it [24].

Several transcription factors are involved in the osteogenic differentiation pathway, including runt-related transcription factor 2 (Runx2), osterix (Osx, or SP7), Smad, and β-catenin [25–27]. Runx2 expressing cells are defined as pre-osteoblasts, a heterogeneous population of cells that includes all cells transitioning from progenitors to mature osteoblasts. A three-stage differentiation of the pre-osteoblasts then follows. The first stage involves cells' proliferation and expression of transforming growth factor-beta receptor 1 (TGF-βR1), fibronectin, collagen, and osteopontin. The second stage involves the initiation of cellular differentiation and maturation of the ECM with alkaline phosphatase (ALP) and collagen expression. In the final stage, the ECM is enriched with osteocalcin, which promotes matrix mineralization [20]. Runx2 guides MSCs differentiation into osteoblasts besides inhibition of other differentiation pathways, particularly adipogenic and chondrogenic ones [28,29]. Various signaling pathways, such as bone morphogenic proteins (BMPs), Notch, and Wnt signaling pathways, could regulate Runx2 expression.

BMPs are famous for their capability to induce bone formation. They activate intracellular Smad, which translocates to the nucleus and acts as a transcription factor besides promoting Runx2 expression [30]. BMP9 stimulates the activation of Smad1/5/8 in MSCs cells. Moreover, Smad4 knockdown decreases the nuclear translocation of Smad1/5/8 and inhibits osteogenic differentiation [31]. Hence, Smad is of great importance, and its interaction with Runx2 is essential for osteogenic differentiation. Mutation of the Cterminal domain of Runx2 disrupts Runx2–Smad transcriptional activities, which leads to the suppression of osteogenic differentiation [32].

Osx is an essential transcription factor for osteogenic differentiation and subsequent bone formation. In Osx null mice, no bone formation occurred; additionally, in the Runx2 null mice, no expression of Osx was noted, indicating that Osx acts as a downstream of Runx2 and emphasizing its role in MSCs osteogenic differentiation and bone formation [33]. Moreover, Wnt signaling pathway activation in MSCs induces Osx expression and suppresses peroxisome proliferator-activated receptor γ (PPAR-γ) [34]. Moreover, β-catenin has a competitive inhibitory relationship with PPAR-γ, where activation of one of them leads to the degradation of the other [35]. Therefore, Wnt/β-catenin signaling activation shifts MSCs' commitment towards osteogenesis at the expense of adipogenic differentiation [34].

β-catenin further plays a critical role in MSCs' osteogenic differentiation. Its absence blocks the osteogenic differentiation and allows for the chondrogenic differentiation of MSCs [36]. Wnt signaling is essential for the β-catenin function. Wnt signaling accumulates β-catenin in the cytoplasm and translocates it into the nucleus, activating the transcription of downstream genes. The absence of Wnt signaling leads to the degradation of β-catenin and interferes with MSCs' osteogenic differentiation [37]. The sensitivity of β-catenin to matrix stiffness during the differentiation of adipose-derived stromal cells (ASCs) has been demonstrated [38]. β-catenin increased nuclear translocation with increased matrix stiffness and enhanced the expression of Runx2, thus stimulating osteogenesis.

Stem/progenitor cells' behavior is largely affected by extracellular signals from the microenvironment, including chemical and mechanical cues from the ECM [39]. Unlike chemical cues, the mechanical properties of the microenvironment act as signals that are consistent along with time and space, thus providing long-range stimulation to cells over long periods and over relatively long distances. Recent literature has focused on the paramount role of the ECM mechanical properties in controlling stem/progenitor cells' behavior, including maintaining their potency, self-renewal and differentiation, migration, proliferation, and interaction with other cells [39,40]. Matrix-related mechanical stimuli, including strain, shear stress, matrix rigidity, and topography, could impact stem/progenitor cell phenotypes through controlling gene transcription and signaling pathways [40,41].

The extracellular-signal-regulated kinase (ERK) and p38 are members of the mitogenactivated protein kinase (MAPK) enzymes family that is concerned with mechanotransduction pathways [42]. ERK is a potent regulator of MSCs' differentiation, as mechanical stimulation activates ERK through integrin focal adhesion complexes and the initiation of MAPK–ERK signaling cascade [43]. Besides ERK, p38 is involved in MSCs' differentiation. The p38-MAPK signaling activity has been identified as an essential factor for osteoblastic differentiation [44–46]. Ras-mediated signaling has been further presented as a master key that affects multiple intracellular pathways, including ERK, PI3K/AKT, and Smad [47,48]. Inhibition of Ras (RasN17) significantly downregulates AKT, ERK, and Smad1/5/8 activation, as well as osteogenic markers' expression. Conversely, active Ras (RasV12) has little effect on osteogenic markers' expression [49]. Consequently, inducing transcription factors to control and guide MSCs' differentiation has become an essential strategy for guided tissue regeneration [26]. Interference between signaling pathways through interaction between different transcription factors can drive MSCs towards specific cell linage; for example, osteogenic signaling can inhibit the adipogenic signaling pathway, and vice versa [41].

Matrix stiffness has a profound impact on MSCs' behavior. The adhesion, proliferation, and spreading capacity of umbilical cord MSCs varied when cultured on polyacrylamide gels coated with fibronectin with different stiffness (Young's modulus: 13–16, 35–38, 48–53, and 62–68 kPa) [50]. Maximum spreading of MSCs was observed with increased matrix stiffness. The soft matrix promoted adipogenic differentiation with high expression of PPARγ and C/EBPα, while MSCs cultured on the 48–53 kPa matrix differentiate into muscle cells with increased expression of MOYG. On the other hand, MSCs cultured on stiff matrix differentiate into osteoblast with increased expression of ALP, collagen type I, Runx2, and osteocalcin [50]. Additionally, bone-marrow MSCs cultured on fibronectincoated polyacrylamide hydrogels with different stiffnesses, ranging from 13 to 68 kPa, demonstrated enhanced adhesion, spreading and proliferation upon increasing matrix stiffness [51]. On 62–68 kPa, MSCs exhibited a polygonal morphology with a more extensive spreading area and high expression of Runx2, ALP, and osteopontin. These data highlight the critical role of matrix stiffness in regulating MSCs behavior which aids in the development of new biomaterials for tissue regeneration.

Insights into how stem/progenitor cells sense signals from the ECM and how they respond to these signals at the molecular level have become an area of increasing research [21,22,52]. Lately, stem/progenitor cells were shown to be capable of sensing and responding to the structural and functional cues of the matrix [22,52], such as the topography of the ECM components, adhesive properties of the ECM, and ECM stiffness [24,53]. The cells adhere to the ECM via several specific cell-surface receptors, known as integrins [21,22]. Integrins transmit signals from ECM to the cells, thus affecting the proliferation and differentiation of stem/progenitor cells through mechanotransduction of signals [21,22]. It is suggested that the cells use actomyosin filaments (stress fibers) contractility for reciprocal interactions with their matrix [23]. When cells are grown in vitro, extensive efforts to mimic the in vivo microenvironments have been made to control and direct stem/progenitor cell commitment into specific cell lineages required for regenerative medicine.

Natural and synthetic polymeric materials could offer versatile matrices, which are biocompatible and biodegradable, with tunable characteristics, precise control of their topography, and ease of processing [54,55]. Biomaterial stiffness, which determines the material's resistance to deformation in response to an applied force, is a vital property in tissue engineering. ECM stiffness is calculated by dividing the load by the elastic deformation of the matrix [56], is denoted by the elastic modulus or Young's modulus (E), and represents the resistance that a cell feels when it deforms the ECM [57].

ECM stiffness guides stem/progenitor cells' differentiation down corresponding tissue lineages [58]. Osteogenic differentiation of MSCs was shown to be favored on more rigid substrate, while adipogenic differentiation is favored on softer substrates [21]. Such control of MSCs fate by matrix stiffness was shown to be complementary to, and even synergistic with, the regulatory effects of specialized cell culture media commonly used to direct mesenchymal stem/progenitor cell differentiation into specific lineages [23]. Various biomaterials coupled with different methods of controlling stiffness are employed to develop specific stiffness ranges for regulating MSCs differentiation in vitro. Controlling of substrates' stiffness could be tuned through adjusting the biomaterial composition, the amount/concentration/ratio of material components, the degree of crosslinking, and the reaction conditions [56]. Taking into consideration that the bulk stiffness of most native tissues is much lower than that of plastic or glassware used for in vitro tissue culture [24], the development of biomimetic polymeric matrices of tunable stiffness, mimicking native tissues, allowed new data to reveal more details on the impact of mechanical cues of the microenvironment, especially ECM stiffness, on cellular properties [58].

In this review, we highlight the regulatory role of matrix stiffness in directing the osteogenic differentiation of MSCs, addressing how MSCs sense and respond to their ECM, in addition to listing different polymeric biomaterials commonly used in vitro and methods used to alter their stiffness to dictate MSCs differentiation towards the osteogenic lineage. Moreover, through the current review, we aim to elucidate the effect of ECM stiffness on the MSCs' osteogenic potential and the underlying mechanism, which is of particular importance during the process of designing new materials for bone-tissue regeneration.
