Mechanical Changes of MSCs and Fibroblasts Figure 6. TCM alters cytoplasmic rheology. The time-dependent ensemble averaged MSDs of 100 nm particles embedded in the cytoplasm of MSCs and 3T3 fibroblasts were converted to frequency-dependent elastic (G9, solid lines) and visous (G0, dashed lines) moduli using a custom algorithm written for Matlab software. The ensemble-averaged frequency-dependent viscoelasticities of MSCs (A–E, left) and 3T3 fibroblasts (F–J, right) prior to (A,F) and 30 minutes (B,G), 1 hour (C,H), 2 hours (D,I), or 3 hours (E,J) after treatment with TCM. The cytoplasm of MSCs became predominantly elastic within 60 minutes; whereas, 3T3 fibroblasts required 3 hours to undergo a similar change. doi:10.1371/journal.pone.0033248.g006 Transwell migration assays further illustrated how crucial this stiffening was for chemotaxis, as significant migration to TCM over control media was not achieved until the time point after cells had transitioned to a primarily elastic phenotype (Fig. 7B). Additionally, in vitro migration assays show that even a one hour exposure to TCM increases MSC migration. Since soluble factors mediate cell migration by interacting with cell surface receptors that require much longer synthesis and transport times [50], these results suggest a novel ‘mechanical priming’ achieved by short- term TCM preconditioning. Initially, the morphology of fibroblasts and MSCs was similar; however, after prolonged TCM treatment, MSCs acquired an elongated spindle-shape morphology, and 3T3 and primary fibroblasts maintained their original shape (Fig. 2C–F). This morphological change in MSCs coincided with the extension of linear actin filaments and microtubules (Fig. 3A). These changes further support the importance of RhoA, as activation of Rho effector mDia1 induces cell elongation coupled with parallel alignment of linear actin and microtubules [51]. The prolonged exposure of MSCs to TCM also resulted in visible loss of stress fibers (Fig. 3B) and focal adhesions (Fig. 4A–C). These changes correlate with increased expression of Cdc42, which can block Rho-induced focal adhesions and stress fibers [52]. Furthermore, Cdc42 is crucial in cell elongation processes, such as neurite outgrowth [53]. ANOVA revealed that only Cdc42 shows a significant interaction effect between cell type and treatment, suggesting it is a primary contributor to the long-term differences. Figure 7. Effect of TCM on Cell Migration. Tranwell assays were used to measure the migration of MSCs and 3T3 fibroblasts through 3 mm- (A) and 8 mm- (B) pore transwell inserts toward CM or TCM. The average number of cells per image (n = 9), collected with a 106-objective, was reported. TCM significantly increased MSC migration, compared to CM, through 3 mm pores within 3 hours and 8 mm pores within 2 hours; however, fibroblast migration was only increased through 8 mm pores within 3 hours. MSCs and 3T3 fibroblasts were then treated with CM or TCM for 1 hour and allowed to migrate through 8 mm-pore transwell inserts toward CM or TCM for 3 hours (C). Pre-treatment with TCM resulted in synergistic effects on chemotactic migration for both cell types. doi:10.1371/journal.pone.0033248.g007 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33248