All body tissues are viscoelastic and undergo creep (or non-reversible

deformation) when put under stress. This is due to the presence of both

elastic and viscous (energy dissipative) components or structures within

tissues. We have previously shown that substrates of constant stiffness

(compressive modulus) but varying viscous properties (which allow these

substrates to display differing magnitudes of creep behaviour)

significantly influence human mesenchymal stem cell (hMSC) behaviour,

promoting differentiation along a smooth muscle lineage, both with and

without the addition of soluble inductive factors. Here, we investigated

the mechanisms behind this effect. We showed that MSCs cultured on high

creep substrates had increased expression of proteins capable of inducing

SMC differentiation, including both soluble factors (TGF-β1 and its

receptor, TGFBR1) and ECM proteins found within blood vessels (collagen

type I, collagen type IV and laminin) as well as N-Cadherin, a cell

adhesion molecule (CAM) that mediates SMC differentiation of hMSCs. This

expression of N-cad was significantly reduced by treating hMSCs with the

Rac1 inhibitor NSC23766 on high, but not low creep substrates, suggesting

that  increased substrate creep causes an increase in Rac1 activity which

subsequently enhances N-cad expression. Supporting this link with Rac1,

hMSC motility and filapodial protrusion rates (both associated with Rac1

activity) were also increased on high creep substrates. This lead us to

the hypothesis that myogenic differentiation of hMSCs on high creep

substrates was enhanced via up regulation of Rac1 activity. To directly

test this hypothesis, a FRET biosensor was used to monitor  Rac1 activity

in C3H10T1/2 mesenchymal cells on high and low creep substrates. This

showed that Rac1 was significantly upregulated in cells on high creep

substrates at both 4hr and 24hr timepoints. Together this work shows that

the smooth muscle differentiation of hMSCs on high creep substrates is

mediated by an increase in Rac1 activity, as well as enhanced expression

of both growth factors and matrix components associated with smooth

muscle differentiation. It is anticipated that the information presented

here will be informative for the future optimisation of biomaterial

scaffolds for tissue engineering applications, such as those involving

blood vessel regeneration