Atomic force acoustic microscopy reveals the influence of substrate stiffness and topography on cell behavior.

Yan Liu,Xing Chen,Meng-Nan Liu,Lu Wang,Zuo-Bin Wang,Li Li,Liang Cao,Jin Yan,Ying Wang

doi:10.3762/bjnano.10.223

Abstract

The stiffness and the topography of the substrate at the cell–substrate interface are two key properties influencing cell behavior. In this paper, atomic force acoustic microscopy (AFAM) is used to investigate the influence of substrate stiffness and substrate topography on the responses of L929 fibroblasts. This combined nondestructive technique is able to characterize materials at high lateral resolution. To produce substrates of tunable stiffness and topography, we imprint nanostripe patterns on undeveloped and developed SU-8 photoresist films using electron-beam lithography (EBL). Elastic deformations of the substrate surfaces and the cells are revealed by AFAM. Our results show that AFAM is capable of imaging surface elastic deformations. By immunofluorescence experiments, we find that the L929 cells significantly elongate on the patterned stiffness substrate, whereas the elasticity of the pattern has only little effect on the spreading of the L929 cells. The influence of the topography pattern on the cell alignment and morphology is even more pronounced leading to an arrangement of the cells along the nanostripe pattern. Our method is useful for the quantitative characterization of cell–substrate interactions and provides guidance for the tissue regeneration therapy in biomedicine.

Highlights

The interactions of cells with extracellular matrices (ECMs) play important roles in regenerative medicine and tissue engineering [1,2] as they affect many cell functions such as cell migration [3,4], attachment, proliferation [5,6] and differentiation [7,8]
From Figure 3m and Figure 3n, we infer that both the elongation and the area spread of the L929 cells on the undeveloped SU-8 films remain statistically unchanged in the stiffness range investigated. These results suggest that modulating the electron-beam lithography (EBL) exposure doses does not significantly influence the adhesion and the spreading of the L929 cells on the patterned stiffness surfaces
The stiffness and the topography of ECMs are important for cell behavior, but there is still a lack of effective methods to characterize these features

Summary

Introduction

The interactions of cells with extracellular matrices (ECMs) play important roles in regenerative medicine and tissue engineering [1,2] as they affect many cell functions such as cell migration [3,4], attachment, proliferation [5,6] and differentiation [7,8]. Substrate stiffness and topography are two of the most important ECM physical parameters in regulating cell. A previous study shows that cells attached to rigid substrates can spread thereby increasing the interaction area with other cells, while cells remain quiescent and are observed to be more spherical when attached to softer substrates [10]. Many native tissues are not homogeneously stiff and it is not clear whether the controlled presentation of rigid and flexible material axes on the substrate governs the cytoskeletal and nuclear morphology [14]

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