Rigidity of silicone substrates controls cell spreading and stem cell differentiation

Abstract

Multiple functions of cells cultured on flat substrates have been shown to depend on the elastic modulus of the substrate, E, with the dependence being strongest in a physiological range of soft tissues, corresponding to E from 0.1 to 100 kPa. Among those functions are stem cell differentiation, cell spreading, and cell signaling [1]. In the context of differentiation of mesenchymal stem cells (MSCs), substrates with E in the ranges of <4 kPa, 8-17 kPa, and >25 kPa, have been classified as soft (adipogenic) [2,3], medium rigidity (myogenic)1, and hard (osteogenic) [1], respectively. In most studies, the soft substrates are hydrogels, and variations in their elastic moduli are usually accompanied by variations in the dry mass and porosity. The paradigm of the effect of substrate rigidity on the cellular functions has been challenged by Trappmann et al. [4], who claimed that cell spreading and differentiation on hydrogel substrates depend not on the elastic moduli of the substrates, but rather on their porosity, which affects the density of adhesion points between the substrate surface and the extracellular matrix (ECM) coating on it. This claim has been rebutted by Wen at al. [3], who have used hydrogel substrates with different porosities but identical elastic moduli to show that it is the elastic modulus rather than the porosity that is key to the effect of the substrate on cell spreading and differentiation. Both publications agree, however, that there is no appreciable effect of the substrate rigidity on either cell spreading or differentiation, if the substrate is made of a silicone gel rather than a hydrogel. This conclusion appears to contradict the findings of several other groups, who reported that when cells are plated on an array of flexible silicone microposts, their spreading and differentiation depend on the rigidity of the substrate [5], and that when cell are plated on silicone gels, their differentiation depends on the gel rigidity [6]. To resolve this contradiction, we used soft, medium, and hard silicone gel substrates with elastic moduli of 0.5, 16, and 64 kPa, respectively, (Fig.1) to perform experiments similar to those reported in Refs.4 and 3, testing the dependence of differentiation and spreading of MSCs and of spreading of fibroblasts and keratinocytes on the substrate rigidity.