Integrins in the Regulation of Mesenchymal Stem Cell Differentiation by Mechanical Signals

Mesenchymal stem cells (MSCs) can sense and convert mechanical stimuli signals into a chemical response. Integrins are involved in the mechanotransduction from inside to outside and from outside to inside, and ultimately affect the fate of MSCs responding to different mechanical signals. Different integrins participate in different signaling pathways to regulate MSCs multi-differentiation. In this review, we summarize the latest advances in the effects of mechanical signals on the differentiation of MSCs, the importance of integrins in mechanotransduction, the relationship between integrin heterodimers and different mechanical signals, and the interaction among mechanical signals. We put forward our views on the prospect and challenges of developing mechanical biology in tissue engineering and regenerative medicine.


Introduction
Mesenchymal stem cells (MSCs) are self-renewable, multipotent, culturally expandable in vitro and with exceptional genomic stability [1]. MSCs are useful in regenerative medicine. The fate of MSCs is affected by many environmental cues, including biological, chemical, and mechanical signals. Mechanical signals are important in development and function.
The mechanical signals for cells can be classified into the internal signals and the external signals [2]. The internal mechanical signals of cells mainly include cellular properties, such as shape, elasticity, and viscoelasticity, and internal forces, such as cell adhesion among cells, and inner cell forces [3]. The external mechanical signals of cells mainly consist of extracellular matrix (ECM) mechanics, such as ECM elasticity and topology, and the external forces. When cells are stimulated by external mechanical signals, the internal mechanical signals will be changed correspondingly [4]. External mechanical forces, such as compressive force, tensile force, fluid shear force and hydrostatic pressure, can also affect the differentiation of MSCs [5].
Integrins participate in many fundamental cellular processes, including survival, apoptosis, proliferation, differentiation and migration by mediating cell-ECM interaction and cell-cell adhesion. The most general feature of integrins is that the interaction of integrins with their ligands can activate inside-out signaling and outside-in signaling [6]. Integrin inside-out activation means the process that activation and subsequent talin, and possibly the binding of kindlin with β-integrin tail change the tilt angle of β-integrin transmembrane domain, and separate α/β-integrin transmembrane domain and cytoplasmic domain, resulting in the Lei Wang and Fuwen Zheng are contributed equally to this study.
The fate of cell is regulated. expansion and opening of outer domain, and finally ligand binding [7]. While outside-in internal activation is that talin and kindlin maintain the rare open integrin state by transferring mechanical forces to the integrin ligand complex, rather than by destroying the transmembrane binding of integrin heterodimers. Thermodynamic equilibrium is the core of this activation model [8].
As cellular function and phenotype are influenced by mechanical signals, the cell-cell and cell-matrix interactions are significant in understanding distinct cellular phenotypes. Integrins play a major role in the mediation of the cell-ECM interactions. Integrins can sense the mechanical signals and activate inside-out and outside-in signaling by interacting with ECM, cytoskeletal and signaling components.

Roles of Different Integrins Heterodimers in Mechanotransduction
Integrins are a family of transmembrane glycoproteins consisting of noncovalent heterodimers. Each family contains a common β-subunit combined with one or more distinct α-subunits non-covalently. All members of the integrin family adopt a shape that resembles a large "head" on two "legs", with the head containing the sites for ligand binding and subunit association. The α and β subunits are constructed from several domains with flexible linkers between them. Each subunit of integrins has a large extracellular region, a single transmembrane domain and a short cytoplasmic tail (except for β4 integrin) (Fig. 1). The N-terminal domains of the α and β-subunits associate to form the integrin headpiece, which contains ECM binding site, whereas the C-terminal segments transverse the plasma membrane and mediate interactions with the cytoskeleton and with signaling molecules [9]. α-subunits consist of four or five extracellular domains, while β-subunits consist of seven extracellular domains [10]. Tails, especially β Chain, which are quite flexible, can form transient structure and the complex composed of the tails and theirs binding protein may provide important information for transduction [9,11]. In vertebrates, there are 18 α and 8 β subunits which can assemble into 24 different receptors with different binding properties and different tissue distribution [9].
At the cellular level, integrins interact with their ligands, such as ECM, by their extracellular region and with signaling components by their intracellular domains. Integrins act mainly in cell-ECM contact sites. The connection between integrin and cytoskeleton is mediated by dynamic integrin adhesion complex, composed of many molecules [12]. Talin and kindlin play regulatory roles in integrin mediated adhesion complex connecting integrin and cytoskeleton. They not only connect integrins directly to actin, but also have multiple regulatory interaction sites related to plasma membrane and other liposome components. In addition, talin protein regulates the strength of integrin adhesion and determines the structure of adhesion sites [13]and mechanical forces can also regulate integrin affinity [14]. The composition of matrix can regulate cells via integrin binding to adhesion sites [15]. Ligand-bound integrins engage the actin cytoskeleton via talin and additional linker proteins, leading to integrin clustering and the ensuing activation of focal adhesion kinase (FAK) and SRC family kinases [16]. Organization Fig. 1 The structure of integrins. Integrins are heterodimers of non-covalently associated α and β subunits. All members of the integrin family adopt a shape that resembles a large "head" on two "legs", with the head containing the sites for ligand binding and subunit association. The α and β subunits are constructed from several domains with flexible linkers between them of the actin cytoskeleton and kinase signaling pathways impinge on Ras-ERK, phosphatidylinositol 3-kinase (PI3K) /AKT, and YAP/TAZ pathways [16]. The interaction of ion channels and integrins are increasingly recognized [17]. Ion channels often appear to coordinate these upstream and downstream signals [18]. Activated K + channels can activate PI3K/AKT pathway and mitogen activated protein kinase module system, including mitogen activated protein kinase 1/2 (or ERK 1/2) [17]. Transient receptor potential melastatin type 7(TRPM7) could independently perceive mechanical stimulation signals and regulate Ca 2+ inflow by giving bone marrow MSCs pressure, thus promoting osteogenic differentiation of human bone marrow MSCs [19]. Integrins can regulate ion channels and form macromolecular complexes [20], though this complex can only form when the channel is closed. Cell response is accompanied by an activation / inactivation cycle of integrin receptor and its channel partners. Terminal differentiation may involve permanent changes in membrane transport [17]. Complex contributes to the localization of the channel onto the plasma membrane. The integrin-channel complex regulates downstream signaling proteins, such as tyrosine kinases and GTPases. After the initial phase of adhesion, the activity of Rho A gradually increases whereas Rac1 activity diminishes. Late adhesion is associated with Rho A-dependent stress fiber formation and focal adhesion maturation [21]. Integrin-channel complex may regulate the differentiation of MSCs by activating GTPases and eventually affect the fiber formation in the cell adhesion process.
There are many studies about the roles of the different integrin heterodimers in mechanical transduction to regulate the differentiation of MSCs (Table 1). The expression of integrin β3 is at different level in MSCs of different shape [22]. Integrin α5β1 is related to internal forces and fluid shear force [23]. MSCs on matrix with titanium are at high expression level of integrin α5β3 and integrin α2β1 etc. [24]. One mechanical signal may also be related to different integrin heterodimers whose correspondence is specific, allowing the corresponding relationship between integrin heterodimers and mechanical signals.
In summary, different integrin heterodimers are conjectured to respond to specific mechanical signals, and the interactions of different mechanical signals are relevant to this kind of respond. Therefore, it is possible to  The external properties include external forces (shear force, compression, tension, hydrostatic pressure), matrix properties (hardness, morphology). While the internal mechanical signal includes internal forces, cell properties (shape, elasticity). Integrin is a "bridge" connecting the internal and external properties of cells, which can respond to external and internal mechanical signals by outside-in and inside-out transduction, and regulate cell differentiation through Wnt, PI3K, MAPK and other pathways, or through the cytoskeleton. Ion channels was thought to play an important role in signal transduction by forming macromolecular complexes with integrins judge whether two or more properties interact with each other according to the change of specific integrins. But it still needs more experiments to support the conclusion and build the corresponding relationship between integrin heterodimers and mechanical signals. The role of integrin in mediating MSC differentiation influenced by internal mechanical signals and external mechanical signals will be discussed below (Fig. 2).

Integrins Affect Stem Cell Differentiation by Sensing the Internal Mechanical Signals
The internal mechanical signals are important in mechanotransduction because the external mechanical signals affect cell behavior eventually by changing the internal mechanical signals [4]. The internal mechanical signals of cells consist of internal forces and intrinsic cellular properties including cell elasticity, hardness, viscoelasticity and especially shape. The effect of cellular properties and internal forces and the role of integrins in the differentiation of MSCs will be discussed below ( Table 2).

Internal Force Affects MSCs Differentiation Through Integrins
Internal forces include forces during cell adhesion between cells and inner cell forces. Forces, from myosin bundles sliding along actin filaments, are transmitted to ECM [25]. Internal forces are produced by myofibril and transmitted by integrin. Integrin can bind to the cytoskeleton of actin, and integrin linked kinase (ILK) can play the role of adaptor protein in the adhesion spot, which can play a role in the binding of cytoskeleton and cell matrix, thus affecting the transduction of Wnt/β-catenin signaling pathway to affect the differentiation of MSCs [26]. ILK has also been shown to regulate the stability and translocation of β-Catenin (an important part of Wnt pathway) in cultured cells [27]. It has been shown that the expression of β-Catenin in T cells can enhance expression of integrin α4β1. While the pharmacological inhibition of integrin α4β1 can reduce the presence of abnormal T cells in the central nervous system of mice expressing β-Catenin [28]. In conclusion, internal forces can affect the binding between integrin and ILK, and then Wnt pathway to determine the fate of MSCs.

Cell Shape Affects MSCs Differentiation Through Integrins
Cell shape results from a force balance inside its mechanical structure [29]. By adjusting their shape, cells can sense the mechanical properties of their microenvironment and regulate traction forces [30]. Cell shape alters the differentiation of MSCs. Round cells promote adipogenesis while high spreading cells prefer an osteoblast fate [31,32]. The round shape induces low contractibility resulting in adipocyte differentiation of MSCs [32,33]. Cell shape determines MSCs differentiation depending on cytoskeletal forces from both the actin cytoskeleton and the microtubule skeleton [34]. 3D cell shape could control integrin-based focal adhesion growth by modulating the level of forces transmitted to the cell environment [29]. The transduction of shape signals is mediated by integrin β3 and its binding partners from the ezrin-radixin-moesin (ERM) family, and activity of integrin β3 affects signal flows from cell shape to Focal adhesions. Focal adhesions assembly and maturation are known to increase with cytoskeletal tensile force, and then the shape signals were decoded by mechanical transduction to activate the Rho pathway. In this process, integrin β3 acts through ERM family to transduce shape signals to the Rho pathway. And talin also plays a role in this process through inside-out signaling and outside-in  [24,26] Internal forces RhoA/ROCK pathway Osteoblast Chondroblast [27,28] Cell-cell adhesion Cadherins Osteoblast [29] signaling [22]. Integrin β3 plays the role in the process that different cell shapes cause different differentiation of MSCs by Focal adhesions, which eventually active the Rho pathway to regulate the differentiation direction of MSCs.

Integrins Affect Stem Cell Differentiation by Sensing the External Mechanical Signals
The external mechanical properties of cells mainly include the matrix mechanics (structure and elasticity of matrix) and the external forces. They are always related to internal mechanical properties. Cell surface integrin receptors sense the biophysical and biochemical properties of the extracellular matrix, convey this information to the interior of the cell, and regulate gene expression during stem cell differentiation [32]. In the process of transduction between external and internal properties, integrin plays an important role (Table 3).

Matrix Mechanics Affect MSCs Differentiation Through Integrins
Matrix mechanics consist of matrix topology, stiffness and so on. Matrix mechanics play an important role in the differentiation of MSCs. Matrix topology influences gene expression and differentiation of MSCs [35]; matrix stiffness can promote the differentiation of MSCs into different linages [36]; the physical properties of the scaffold can also determine MSC differentiation [37]. And cell-matrix interaction mediated by integrins can activate specific signaling pathways.

Matrix Topology Affects MSCs Differentiation Through Integrin
The topological structure of matrix plays an important role in regulating cell behavior, such as affect the differentiation of MSCs by affecting the mechanical properties and metabolic expression of MSCs. Cells are surrounded by a complex microenvironment, encompassing both cellular and ECM components [38]. ECM topological structure is a general concept of the morphology of cell living microenvironment [38], including size, shape, and geometric arrangement etc. Matrix topography is a powerful mean by which MSCs activities can be modulated [39]. They can exert strong effects on many cell behaviors such as adhesion, migration, alignment, and differentiation. Among these factors, size of topography (such as width, spacing, and depth of features) appears to play a crucial role in modulating cell behaviors.
And microtopography (such as feature size larger than 10 μ m, which is the length-scale of a mammalian cell mainly has effects on the whole cell shape, whose role in differentiation was discussed before [38]. Topography in microstructure regulate MSCs fate [40]. Micro-grooved topography can directly regulate MSCs morphology and expression of the contractile structural maintenance of chromosomes (SMC) protein markers [41], and possesses the potential to directly enhance MSCs mechanical properties [39]. In vitro, the morphology of micropores /microspheres coated with silicon on the surface of titanium significantly enhanced the initial adhesion, proliferation and osteogenic differentiation of MSCs [42]. Microtopography increases osteogenic differentiation by increasing the expression of lncRNA PWRN1-209, which may be regulated by the signal of integrin-Focal Adhesion Kinase (FAK)-alkaline phosphatase (ALP) [43].
2D and 3D structures have different effects on the differentiation of MSCs. When human adipose-derived MSCs was implanted into a 3D culture system lacking bioactive substances (globules and polystyrene scaffolds), the expression of osteogenic markers was higher than that of 2D culture. Compared with 2D culture, the differentiation of adipocytes decreased. Finally, the cells were dissociated and reprecipitated in 2D culture medium of osteoblasts to induce late differentiation. After the next 14 days of maturation, the cells produced bone minerals and osteocalcin proteins, which were late markers of osteoblasts. This suggested that 3D environment may provide additional stimulation for adipose-derived MSCs to osteogenic differentiation [44]. In addition, the surface structure of the matrix also affects the differentiation. The pore size of the coated surface also affected the adhesion and osteogenic differentiation of MSCs. In the range of 3-10 μm, larger pore size was more favorable for the adhesion and osteogenic differentiation of MSCs [45].
The effect of ECM topology on the differentiation of MSCs is related to integrin. MSCs have to establish appropriate contacts with ECM which is mainly composed of collagen type I, IV and fibronectin in order to differentiate into cells with a specialized function. Integrin is one of the most important receptor families mediating cell-ECM interactions. Integrins do not only establish cell bonds with a range of ligands but also regulate cytoskeletal dynamics and initiate various signals that are essential for the regulation of cellular processes, such as cell differentiation [46].
Matrix topology affects osteogenic differentiation of MSCs by integrins. The micro topological structure with titanium of ECM can induce osteogenic differentiation of cells by integrin β3 [47] and αV [48]. And osteoblasts cultured on microstructure titanium need to send out signals through α2β1 to produce soluble paracrine autocrine factors that participate in the differentiation of MSCs [24]. Knockout of β1 or α2 integrin subunits in osteoblasts can weaken the ability of osteoblasts to recognize complex microstructure titanium surface, resulting in cell failure to differentiate or produce osteogenic environment [49]. And integrin α2β1 can promote osteogenic differentiation [43]. Rough surface topography promotes osteogenic differentiation by Wnt/β-catenin signaling pathways [50]. Integrins specifically participate in process that matrix topology affects MSCs differentiation by mediating cell-ECM  [15,16] interactions, both integrin α2β1 and αVβ3 promoting osteogenic differentiation of MSCs.

Stiffness/Elasticity Affect MSCs Differentiation Through Integrin
Stiffness is one of the commonly used indexes to evaluate the mechanical properties of materials. According to GB/T 231.1-2018, Brinell stiffness was proportional to the quotient of the test force divided by the indentation surface area. Elasticity refers to the property that an object can regain its original size and shape after deformation. Under the assumption of linear elastic behavior, matrix stiffness was often synonymous with elasticity [51]. Matrix stiffness can affect the morphology, adhesion, proliferation, multipotency and differentiation of MSCs [52]. Soft substrates (1.5 or 15 kPa polydimethylsiloxane) maintain the pluripotency of MSCs. MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes [53]. MSCs are more likely to differentiate into endothelial cells when matrix stiffness is low [36]. Soft scaffolds favored chondrogenic differentiation, while hard scaffolds initially favored osteogenic differentiation [54]. Integrin β1 play a vital role in MSC sensitivity to stiffness [55]. The expression of osteogenic genes on the matrix with high elasticity is higher, which can promote the osteogenic differentiation of MSCs, while the matrix with low elasticity can promote adipogenesis [56]. In the changing process of matrix stiffness from soft to hard, the osteogenic differentiation of MSCs was inhibited in the beginning, but finally promoted osteogenic differentiation in a long time [57]. In contrast, although the softening microenvironment (from hard to soft) finally showed lower osteogenic differentiation than the harder matrix, MSCs still retained the early strong osteogenic differentiation [57]. With the improvement of ligand adhesion and matrix stiffness, cell proliferation and chondrogenic differentiation were enhanced [58]. High stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies [59]. Gene-activated matrices with optimized mechanical properties can induce cartilage formation [59]. Matrix stiffness (62-68 kPa) promotes the osteogenesis of MSCs with high expression levels of Runt-related transcription factor 2 (Runx2), ALP and osteopontin, and integrin α5β1 plays an important role [52,60]. Stiffness is a passive mechanical parameter, which cannot be directly perceived by cells. In order to detect matrix stiffness, cells need to actively use their cytoskeleton to deform the surrounding ECM through integrin bonds. The force generated will depend on the stiffness of ECM, cell will judge its stiffness according to the higher or lower force generated by the surrounding matrix.
In conclusion, matrix stiffness can promote the differentiation of MSCs into adipocyte, endothelial cells, cardiomyocyte, myocyte, chondrocyte, and osteoblast [61]. Weak cell adhesion strength on the soft substrate may be one of the contributing factors which can limit the force applied to the substrate by the cells, therefore modulate cellular transcriptional activities, and then affect the differentiation of MSCs [62]. Cells with different integrins (α5β1 and αVβ6) of different mechanical properties and show different stiffness thresholds in mechanical sensing [63]. Matrix stiffness (62-68 kPa) promotes the osteogenesis of MSCs through integrin α5β1 [52,60]. Matrix stiffness and interleukin-1 (IL-1) beta activate the ERK1/2 signaling pathway and promote osteogenic differentiation. The p38 signal activated by IL-1 beta plays a strong role in inhibiting osteoblast differentiation, thus weakening the important role of ERK1/2 signal. Activation/inactivation mechanics of sensitive factors (such as FAK, ERK, p38) also plays a key role in the synergistic effect of matrix stiffness and IL-1 beta on osteogenesis of MSCs [64]. In addition, soft substrates (1.5 or 15 kPa polydimethylsiloxane) maintain the pluripotency of MSCs. MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes [53]. As mentioned above, cells on soft substrate may have weak cell adhesion strength, which can limit the level of force applied to the substrate by cells and therefore modulate cellular transcriptional activities. Integrin plays a key role in cell adhesion in mechanical transduction and chemical regulation.

Coupling of Stiffness and Topology
ECM physical properties, including stiffness and topography dominate the fate of stem cells together. For instance, MSCs display characteristics of neurogenic, myogenic, and osteogenic phenotypes after being cultured on hydrogel substrates mimicking the stiffness of neural, muscle, and bone tissues, respectively. And different sizes of fibronectin "island" can restrict MSCs spreading and determine their differentiation. The flatten MSCs, which adhere and spread, undergo osteogenesis, but the round MSCs undergo adipogenesis [65]. These factors usually do not affect cell differentiation alone but are combined by the coupling of stiffness and topological structure. Topography interacts with stiffness and culture time to regulate cell viscoelasticity and gene expression [39].
MSCs respond to surface roughness in relation to stiffness by reorganizing the hierarchical structure of the surface. On extremely soft hydrogels (3.8 kPa) with very high surface roughness, the mechanical response and osteogenesis of the cells were significantly enhanced, which was comparable or even better than the response on smooth and hard substrates [66]. The arranged fiber matrix synthesized by mouse embryo osteoblast precursor cells, MC3T3 cells, was used as the acellular matrix to promote the proliferation of MSCs. Fiber arrangement and matrix stiffness together determine the fate of cells. The soft matrix retains the characteristics of stem cells, while the hard matrix induces differentiation. Well-aligned soft matrices promote spontaneous adipose differentiation, while well-aligned hard matrices reduce crossdifferentiation [67]. The effects of topological structure, surface roughness, alignment and matrix stiffness on the differentiation of MSCs were related to integrin-mediated cell adhesion [40].

External Forces Affect MSCs Differentiation Through Integrins
External forces are a series of forces acting on cells. Hydrostatic pressure, tensile force, fluid shear force and compression etc. all show ability to affect the differentiation of MSCs. And integrin plays an important role in the regulation process.

Hydrostatic Pressure Affects MSCs Differentiation Through Integrin
Hydrostatic pressure can promote osteogenesis and chondrogenesis of MSCs [68]. Hydrostatic pressure is the pressure produced by liquid, and physiological hydrostatic pressure is the pressure exerted on the surrounding tissues by the liquid accumulated in one part of the body. It is the force exercised by the surrounding fluid to cells membranes. Due to its nondirectional nature, it is mainly non-deforming but has an important thermodynamic effect on the cytoskeleton influencing microtubule stability [69]. The integrin α5β1-FAK signaling pathway is involved in regulating biological responses to mechanical stimuli under hydrostatic pressure [70]. In the medium of agarose and fibrin, periodic hydrostatic pressure decreased the activity of alkaline phosphatase, indicating that hydrostatic pressure played a key role in maintaining chondrogenic differentiation by changing the activity of alkaline phosphatase [71]. The expression of osteogenic differentiation genes in MSCs stimulated with a reactor capable of generating perfusion and fluid power in the scaffolds under the hydrostatic pressure of 60 mm Hg (7.98 kPa) increased [72]. When physiological range pressure (0.55-5 MPa at 1 Hz; 4 h/d) was applied to MSCs using a loading regime, there is an increase in glycosaminoglycan and collagen matrix synthesis, which is with respect to the unloaded control [73].
Extracorporeal circulation hydrostatic pressure can promote the osteogenic response of MSCs, and the osteogenic response is stronger with the enhancement of stimulation. Cyclic hydrostatic pressure is an important inducement for cytoskeletal recombination and enhanced osteogenic differentiation of MSCs. Low internal circulating hydrostatic pressure (2 Hz and 10 kPa) in bone can promote the formation of osteogenic lineage of MSCs, and higher hydrostatic pressure of 300 kPa stimulates a stronger osteogenic response [74]. The expression of integrin β5 increases after hydrostatic pressure stimulation, which may inhibit osteoclast maturation, promote differentiation, and enhance migration during hydrostatic pressure-driven osteogenesis in vitro [75]. Moreover, the intermediate filament network undergoes breakdown and reorganization under hydrostatic pressure, which is required for loading induced MSCs osteogenesis. And bundled centripetal intermediate filaments translocate toward the perinuclear region [76]. Anthrax toxin protein receptor (ANTXR)1 plays a key role in chondrogenesis of MSCs under hydrostatic pressure, and ANTXR1 is a novel mechanosensor on the cell membrane, acting independently from the classical mechanoreceptor molecule integrin β1 [77]. High expression of integrin β5 promotes osteogenic differentiation under hydrostatic pressure force and plays a main role in the process which is possibly related to intermediate filaments.

Tensile Force Affects MSCs Differentiation Through Integrin
Within the elastic range, the deformation caused by the external force is proportional to the external force. Deformation varies according to the direction in which the force is applied, and the force that causes the object to extend is called tensile force. Tensile force plays role in determining the fate of MSCs. Tensile force refers to the external stimuli that tends to stretch cells, acting in opposite directions, thus causing their elongation. Cellular responses to stretching depend largely on the type and amount of load as well as on the composition of the ECM [69]. Tensile force has been shown to affect a number of physiological processes in MSCs, including cell movement, differentiation, and histogenesis. At present, it has been confirmed through experiments that tensile force can enhance the expression of fibrosis [78], ossification [79] and chondrogenic [80] genes of MSCs. Tensile force increased the expression of integrin β1 in MSCs. Integrins provide a preferred site for mechanical signal transfer across the cell surface and transmit the signal from the plasma membrane to the cytoskeleton [81]. It has been confirmed that uniaxial or biaxial tensile force can promote chondrogenic differentiation of MSCs [82]. Applying 10% radial tensile force to bone marrow MSCs can induce their differentiation into fibrochondrocytes of temporomandibular joint disc [83]. Cyclic stretching can promote chondrogenesis of bone marrow MSCs by upregulating the expression of miR-365 [84]. Furthermore, cyclic stretching can improve alkaline phosphatase activity and promote osteogenic differentiation of bone marrow MSCs [85]. Proper tensile stimulation can improve the viscoelasticity and chondrogenic phenotype of MSCs [86]. In addition, when co-cultured with appropriate cyclic sinusoidal dynamic tensile force, the proliferation capacity and cartilage phenotype of the bone marrow MSCs and chondrocytes were improved [87].
The balance of the expression of β1 and β3 integrins determines the homeostasis of intracellular tensile force. The loss of β1 integrin leads to the decrease of contractile force, while the deletion of β3 integrin led to the increase of β1 integrin activation. These result in changes in cell shape and spatial distribution of cell traction [88]. When using selfassembled block copolymer surfaces to present nanodomains of an adhesive peptide found in many ECM proteins at different lateral spacings (from 30 to 60 nm), MSCs on smaller nanodomain spacings suffered higher tensile force produced larger, longer-lived, and increased numbers of Focal adhesions because more activated FAK and Src proteins were recruited compared to those observed on larger nanodomain spacings. Besides the expression of Rac1, β-catenin and the nuclear localization of YAP/TAZ and RUNX2 on smaller nanodomain spacings were at high level, which together biased the commitment of MSCs to an osteogenic fate [89]. Integrin-β1 mediates osteoblast differentiation and osteoblastic ECM formation are promoted by cyclic tensile forces. 5%-10% strain amplitude cyclic sinusoidal dynamic tensile mechanical stimulation increases glycosaminoglycan and type II collagen expression to improve the cartilage phenotype of chondrocytes [87].
In summary, uniaxial or biaxial tensile force and cyclic tensile force can promote chondrogenic differentiation of MSCs. Cyclic tensile force also promotes osteogenic differentiation of stem cells. Integrin β1 and β3 are involved in the regulation, which can determine the homeostasis of intracellular tensile force, and regulate the nuclear localization of YAP/TAZ and RUNX2.

Fluid Shear Force Affects MSCs Differentiation Through Integrin
According to the definition of material mechanics, "shear" is the relative staggered deformation of the cross section of a material under the action of a pair of closely spaced of the same size and pointing to the opposite lateral external force. The force that causes the material to produce shear deformation is called shear force. When two opposite forces were tangentially applied to cells surface, they generate shear stress, which caused changes in morphology and adhesion properties [69]. One model for the role of integrins in shear stress signaling is that tension generated at the apical surface is transmitted through the cytoskeleton to integrins localized to the basal surface, thereby inducing structural changes that enhances their affinity for ECM ligands [90]. Rho dependent contractile protein and fluid shear force could affect the osteogenesis of cells [91]. A quantitative study on the morphological changes of cells receiving fluid shear force showed that cytoskeleton is very important in the response of cells to fluid shear force. With the increase of fluid shear force amplitude, the aggregation of cytoskeleton became more obvious. The response of osteoblasts to fluid shear force may be related to extracellular calcium [92]. Activation of α5β1 integrins by shear stress leads to Ca2 + signaling [90]. Other studies have shown that controlling fluid shear force on substrates with different topography can change the contraction force of cells and thus affect the differentiation of MSCs [93].
Linear shear regulation improves vascular graft retention of fat stem cells by raising integrin α5β1 [23]. Rho ROCK pathway mediates high tractive force under high shear stress, and the activation of integrin that responds to shear stress leads to downstream effectors such as Rho RAC to affect the contractile force of cytoskeleton, and may lead to changes in cellular response [88]. Under fluid shear force, integrin α5β1 affect osteogenic differentiation, aggregation of cytoskeleton and contraction force of cells may be related to activation of Rho ROCK pathway.

Compression Force Affects MSCs Differentiation Through Integrin
The compression force is the value of the compression load applied to the specimen during the compression test divided by the original cross-sectional area of the specimen. Contrary to tensile force, compressive forces applied from the outside towards the center of cells result in cell contraction and shortening [69]. The regulation of compression force could promote the change of chondrogenic gene expression in bone marrow MSCs without the induction and intervention of exogenous growth factors, indicating that compression force is an important factor regulating the differentiation of bone marrow MSCs [94]. Mechanical loading activates FAK and ERK/MAPK pathways via binding of components of the extracellular matrix (ECM) to integrins on the cell surface [95]. Compression force would affect the expression of the transforming growth factor (TGF-β), thus affecting chondrogenic differentiation of bone marrow MSCs [96]. Compression force not only promote MSCs into cartilage differentiation by enhancing cartilage ECM synthesis [97], but also suppressed the hypertrophic development [98].
The crosstalk of TGF-β/SMAD and integrin signaling is important in regulating the compression-driven hypertrophy chondrogenesis [99]. By using MSCs-collagen microtissues as a 3D model, upon short-term dynamic compression, integrin αV binding, focal adhesion formation, FAK activation, and YAP activation are stimulated. integrin αV binding, focal adhesion formation, and subsequent FAK activation, are stimulated [100]. More importantly, long-term compression induces maturation of α5-integrin based adhesions to form long, slender 3D-matrix adhesions, and potentiates osteogenesis [100]. Collectively, compression can modulate cell-matrix interactions significantly mediated by integrins in a dynamic manner, and affects cell fate decisions.

Microgravity in Spaceflight Affects MSCs Differentiation Through Integrin
Microgravity is a state in which the gravitational force acting in any single direction is negligible. Space microgravity decreased the expression of genes specific for osteogenesis and played a dual role by decreasing RUNX2 expression and activity through integrin/FAK/ERK pathways and increased the expression of genes specific for adipogenesis [95]. Microgravity in space flight can reduce bone formation which is related to the osteogenic differentiation of bone marrow MSCs. In an experiment of cell culture on sj-10 satellite, MSCs were induced to differentiate into osteoblasts for 2 and 7 days under the condition of osteogenic induction. However, because of space microgravity, the expression of osteogenic markers decreased, while the expression of 4 lipogenic genes increased, indicating that space microgravity inhibited osteogenic differentiation and resulted in lipogenic differentiation [95]. In endothelial cells, microgravity plays an important role in regulating cell adhesion, actin filament arrangement and tube formation [101]. Gravity destroys the adhesion ability of stem cells, but it can also drive cell differentiation and undifferentiation. Microgravity improves the differentiation ability of stem cells by up regulating hepatocyte specific albumin and cytokeratin 18. In space, the expression of integrin β1, β-actin, α-tubulin and mechanosensitive molecules of Ras homologous gene family member is increased, while the expression of cell surface glycoprotein CD44, intercellular adhesion molecule 1, and Rho related helix coil kinase decreased. Depolymerization of actin filaments and accumulation of microtubules and vimentin were also observed in space by changing the expression and location of focal adhesion complex, Rho guanosine 5 '-triphosphatase. And enhanced exosome mediated mRNA transfer [102].The expression of integrin β1 and relevant proteins promoted by microgravity in spaceflight cause the inhibition of osteogenic differentiation and the promotion of lipogenic differentiation.

Relationship Between Different Mechanical Signals
After introducing the effect of internal and external mechanical properties in the differentiation of MSCs, and the integrin's role in the regulation process separately, the relations of these properties in the regulation of MSCs should also be concerned.
Integrin is the "bridge" between cytoskeleton and ECM, which is pulled by both of them to achieve balance. The properties of extracellular force and ECM need to be transferred to the cytoskeleton through ECM through integrin, and the process of integrin to cytoskeleton transfer is carried out through adaptor proteins. Although it is not clear how many adaptor proteins distribute force, there is no doubt about the pivotal role of integrin in it [63].
Various extracellular forces act on cells to stretch or compress them. Integrins will be activated and aggregate to form adhesion complexes, that is, adhesion maturation, and then affect downstream signaling molecules, and ultimately affect nuclear transcription [63]. The process of activation and signal transduction is related to the "insideout" and "outside-in" signal transduction of integrin, which depends on the conformation change of integrin extracellular domain and the binding of integrin α and β domains [103] and binding of integrin cytoplasmic tail and ILK [104].
Mechanical signals can interact with each other. External forces can stretch or compress cells and then be sensed by cells. And this process is mediated by integrin. Integrin binding and cytoskeletal reorganization are all involved in mechanotransduction, none of these factors in isolation was able to completely explain the temporal mechanosensitivity of MSCs [105]. When suffering forces, integrin can reinforcement which is mediated by capture bond or mediate adhesion mature which raise integrin-ECM adhesion to make a "bridge" between the ECM and cytoskeleton. Cohesion protein in adhesion complex paly roles in the process [63]. Matrix topology can affect the cell shape, thus can change the internal forces to change the open state of integrin. And external forces like tensile force are also influenced by cell adhesion, thus can affect the connection between integrin and cytoskeleton by integrin adhesion complex. And external forces can influent the internal forces by changing the cellular properties like cell shape, which is associated with integrin signaling transduction. There are also external nondirectional forces affecting the matrix properties. Matrix density and/or stiffness which modulate the development of the pericellular matrix, consequently integrin binding and cytoskeletal structure influence the response of MSCs to compression [105]. And bone induced demineralization bone matrix scaffolds have the characteristics of chondrogenesis under the condition of hydrostatic pressure of extracorporeal circulation [106]. Matrix properties can regulate the integrin. Study shows that MSCs differentiated more dominantly into osteogenic cells and predominantly into adipogenic cells on fibronectin which support integrinmediated adhesion than on maltose-binding protein-fused basic fibroblast growth factor which restrict integrin-mediated adhesion by actin cytoskeleton organization and focal adhesion kinase phosphorylation [107].
Internal forces are important in the regulating process of external properties. The forces produced by the cytoskeleton can detect the mechanical properties of the ECM. In turn, this affects the tissue and cellular behavior of the cytoskeleton [88]. Matrix properties can affect the cytoskeleton by changing the FA, and the changing of internal forces can also pass to the ECM through integrin. That's external-internal and internal-external activation.
Many different pathways are in common in the mechanotransduction process. A typical example is Wnt pathway involved in many physical and pathological processes. Rough matrix topology promotes osteogenic differentiation related to Wnt/β-catenin signaling pathways [50]. And internal forces are related to Wnt pathway through integrin and ILK [26]. In addition, tensile force is related to the formation of ECM, and ECM is an important factor in Wnt pathway, so tensile force may also affect Wnt pathway. Rho pathway also plays its role in the adhesion. Rho GTPases play an important role in regulating the downstream cell diffusion, adhesion and migration after integrin binding to ECM [21]. Therefore, Wnt pathway is common in the differentiation regulation of tensile force, internal forces and matrix topology, mainly induced by ECM and β-catenin (Fig. 3).

Outlook and Concluding Remarks
Throughout the lifespan, increasing physical activity positively affects physical health. It is generally believed that mechanical loading changes skeletal muscle pressure and tension, and the physical activity signals are then translated into cells to influence metabolism and structures. Integrins can transmit the mechanical signals, and eventually affecting the fate of MSCs. The cell senses the external forces by cellular mechanical properties, and the matrix mechanical properties by integrins and cytoskeleton. The internal mechanical forces will change according to other mechanical signals. The common pathways exist in different mechanotransduction process. But some specific regulatory mechanism of mechanical signals and the role of integrins are still not very clear. Therefore, in future research, we should further explore the regulatory mechanisms from the view of mechanical properties and external mechanical forces, figure out the role of integrins during mechanotransduction process, and reveal the molecular mechanisms directly related to mechanical signals to promote the clinical application of stem cell therapy and the development of tissue engineering and regenerative medicine.
The mechanical environment is complex in the process of embryonic development and diseases. All kinds of mechanical signals interact with each other. Integrins play an important role in signal transduction. However, to promote the clinical application of stem cells, the way to build a complex, interactive and mutually controlled mechanical environment is still a difficult problem. Knowing how different integrin heterodimers are related to different mechanical signals can help solve it, which may require knowledge related to mechanics, mathematics and computer technology to build a corresponding relationship between mechanical signals and integrin heterodimers. Fig. 3 Relationships of internal and external properties and the activation of integrins. External properties control cell fate through internal properties, and internal and external signals interact and correlate with each other. Under the action of external forces, integrin can enhance the adhesion between integrin and extracellular matrix through capture bond mediated enhancement or mediated adhesion maturation, build a "bridge" between extracellular matrix and cytoskeleton, and finally affect cell behavior through matrix. External forces can affect the properties of matrix, such as hydrostatic pressure can affect the properties of matrix by affecting cell behavior, and ultimately affect cell differentiation (a). In addition, the external forces can also affect the properties of cells, such as under different tensile and compressive forces, cell shape will have different changes (b). The perception of matrix properties is that the cells squeeze the matrix through the cytoskeleton to deform the matrix and produce a force on the cells, which is transmitted into the cells through the cytoskeleton to change internal force, and then the cells produce different reactions according to the force (c). Cell properties such as cell shape can regulate cell behavior by changing internal forces and activating internal mechanical signaling pathways (d). Inside-out: Activator (such as internal forces) induced activation and subsequent talin, and possibly the binding of kindlin with β-integrin tail change the tilt angle of β-integrin transmembrane domain, and separate α/β transmembrane domain and cytoplasmic domain, resulting in the expansion and opening of outer domain, and finally ligand binding. Outside-in: the binding of integrin and its various ligands induces "external" signals on the membrane, enabling cells to sense the extracellular environment and respond accordingly. In addition, in the process of mechanical activation, talin and kindlin maintain the integrin open state by transferring small mechanical force to the integrinligand complex