For decades, neuroscience has been primarily focused on the roles of the molecular and chemical signals that regulate neurogenesis. There is a growing body of evidence that indicates the importance of force and mechanical determinants in neurogenesis. The mechanical rigidity of the extracellular matrix, in particular, has been identified as a regulator for the neurogenesis and lineage commitment of neuronal stem cells. Recent in vitro studies have demonstrated that neuronal outgrowth in 3D strongly depends on the mechanical stiffness of the surrounding matrix. More specifically, it has been shown that the growth and lineage commitment are promoted when the neuronal stem cells are surrounded by a soft 3D matrix, with a Young’s modulus less than E = 5 kPa. The reported results, however, are conflicting when neurons are cultured on 2D soft substrates. While some studies indicate neurons show a bias for growth on softer substrates, others reported that the neuro and dendrogenesis directly correlate with the substrate stiffness. To resolve this discrepancy, in this study, we cultured rat hippocampal neurons on flexible polyacrylamide substrates coated with fibronectin. Polyacrylamide gels were polymerized with crosslinking of acrylamide and bis-acrylamide. A transition in rigidity was introduced in the central region of the substrate by changing the concentration of bis-acrylamide crosslinker. This way, the growth rate of neurons could be compared between the soft (E 50 kPa) regions of the same substrate while all other experimental conditions are similar for the two regions. We hypothesize that the conflicting results previously reported for rigidity sensing of neuronal stem cells on 2D substrates could be rooted in different experimental conditions provided for cells growing on soft and rigid substrates.
Phillips et al. (Sun,) studied this question.