Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells

Abstract

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Figure 1

(A) Scanning electron micrographs of gratings with 350nm linewidth and 700nm pitch (350nm gratings), and 500nm linewidth and 1μm pitch (500nm gratings) fabricated by soft-lithography and heat embossing on PDMS and tissue culture polystyrene (TCPS), respectively. (B) Atomic force micrograph (AFM) of 350 nm gratings on TCPS.

Figure 2

Densitometeric analysis of integrin expression at the cell-matrix interface in human mesenchymal stem cells (hMSCs) cultured on heat-embossed TCPS with 350nm gratings (T-350) or unpatterned control (TCPS), or PDMS with 350nm gratings (P-350) or unpatterned control (PDMS).

Figure 3

(A) F-actin cytoskeleton visualized by Oregon-green labeled phallodin in hMSC on PDMS with 350nm gratings or unpatterned PDMS. (B) Distribution of focal adhesions visualized by immunofluorescence staining of tyrosine-397 phosphorylated FAK (pFAK, red) and F-actin (green). (C) Distribution of focal adhesions visualized by immunofluorescence staining of vinculin (red). Grey boxes indicate the area of the magnified views shown in the insert figures; bar = 10μm in the insert figures. Images of (A) are taken with fluorescence microscopy; images of (B–C) are taken with confocal microscopy.

Figure 4

(A) Expression of focal adhesion-associated proteins and cytoskeletal proteins on Western Blotting. (B) Densitometric analysis of the protein expression, which is normalized to the GAPDH loading controls. (C) Phosphorylation level of FAK in hMSCs cultured on different surfaces.

Figure 5

Mechanical properties of hMSCs cultured on different surfaces as measured by AFM indentation. Results indicate a dramatic effect of the surfaces on the elastic modulus (E_elastic), relaxed modulus (E_relax), instantaneous modulus (E₀) and apparent viscosity of attached cells. (p<0.05 for all the groups not connected by the brackets)