How Deep Might Myoblasts Sense: The Effect of Substrate Stiffness and Thickness on the Behavior of Myoblasts

Mechanical characters of extracellular matrix, such as the stiffness and thickness, have been shown to impact an abundance of cellular processes, including cell spreading, adhesion, proliferation and differentiation. In this study, we used polydimethylsiloxane (PDMS) films of variable thickness and stiffness to investigate the impact on skeletal muscles cell (C2C12 cells) behavior, in more detail. Furthermore, we utilized, for the first time, a heat sensitive material, poly-[N-isopropylacrylamide], in the process of film thickness measurement to obtain more complete films. Results confirmed that C2C12 cells grow better on stiff substrates. Also, our research demonstrated that film thickness has an influence on C2C12 cells attachment and growth. Specifically, when the elastic modulus of the substrate was 5 kPa, cells seeded on thin gels (h < 38 μm) were found to establish large, well-organized and well-spread focal adhesions. In addition, an increase in proliferation can be observed when the gels were 18 μm or thinner. The differentiation of C2C12 cells was also influenced by gel thickness. Myotubes formed on thick PDMS films (h > 38 μm) were generally differentiated by single myoblasts. When growing on thinner gels, myotubes appeared more elongated and multinuclear. Moreover, sarcomeres began to form when cells were seeded on substrates of 38 μm (or less). However, when the elastic modulus was 1.72 MPa, altering the thickness of the PDMS films had no significant impact on spreading, adhesion or proliferation. In short, we conclude that C2C12 cells are able to sense the underlay when growing on a stiff or a thin (h < 38 μm) substrate, which is reflected in their development.