An ELP-based recombinant protein has been produced to develop a bioactive matrix that can stimulate the neuronal cell behaviors and differentiation. To evaluate biological effects of RGD-modified ELP on neural cell behaviors and particularly on neural differentiation in vitro, bioactive substrates were prepared by physically adsorption of the RGD-modified recombinant ELPs onto the conventional TCPS. Isothermal adsorption process has been known as a robust method to generate biomimetic surfaces suitable for displaying and investigating the effects of ECM functional domains on neural differentiation .
Cell adhesion affinity of [RGD-V6
20 was approximately 2-fold higher than that of [V7
20. The mode of N2a binding to [RGD-V6
20 followed hyperbolic saturation and was similar to the adhesion of the cells on fibronectin-coated surface, where cell attachment reached a plateau at the high surface density of the RGD peptide. Both the enhanced attachment of the cells onto [RGD-V6
20 in comparison with that on [V7
20 and saturable binding mode are indicatives of RGD-mediated cell binding through cell-surface receptors. Based on the numerically estimated saturation values, the difference in maximum cell adhesion capacity between [RGD-V6
20 and fibronectin would fall within 2 orders of magnitude. N2a cells were found to have a higher affinity for [V7
20 than for TCPS control surfaces. This is similar to the enhanced interaction observed between fibroblasts and X20-poly(GVGVP), but only if FBS was added to the assay media . However, throughout this study, [V7
20 was inefficient in stimulating N2a cell migration and differentiation, indicating that the interaction of N2a cells with [V7
20 is physiologically irrelevant. Therefore, attachment of N2a cells to the [V7
20-coated surfaces would possibly be mediated by cell-adhesion molecules originated from the FBS, as shown by the fibroblast adhesion to the poor-cell-binding X20-poly(GVGVP) .
The effect of [RGD-V6
20 coatings in N2a cells spreading experiments showed a similar cellular morphology to that seen on fibronectin-coated surfaces, which seems to correlate with enhanced cell spreading. The result presented in this study is in good agreement with previous report whereby fibroblasts formed spreading structures such as lamellipodia and filopodia at the edges of the cells grown on the [RGD-V6
20 matrix .
On the cell-adhesive surfaces, cell locomotion is governed by the interactions between the integrin receptors and their ligands displayed on the cell surfaces . In comparison with the motility on [V7
20, which lacks integrin-adhesion ligands, N2a cells migrated much faster on [RGD-V6
20, and therefore, this was attributed to the RGD-mediated cell adhesion. Until 24 h of culture, the number of cells that migrated across the scratched edges was not statistically different among all 4 substrates. However, during the 24–30 h culture period, a significant increase in the population of migrating cells was measured on both [RGD-V6
20 and fibronectin but not on TCPS and [V7
20. The result suggests a 2-phase migration mode, that is, initially slow movement followed by a fast migration stage, and also implies that [RGD-V6
20 activate signaling events within the cells to express a motile phenotype during the slow migration period.
The incorporation of RGD motifs into the relatively inert elastic VGVPG backbone has yielded an ECM analogue with fibronectin-like function in directing neural differentiation. For instance, immunofluorescence analysis of Tuj1 expression showed that [RGD-V6
20 promote neuronal differentiation along with neurite extension even without RA treatment. Unlike [RGD-V6
20, the [V7
20 was not able to promote neurite elongation, and the lack of neurite outgrowth on the matrix is possibly due to the absence of the RGD motifs that are necessary for proper neurite elongation . Thus, enhanced neurite elongation from N2a cells reflects the activation of signal transduction at the N2a cell-[RGD-V6
20 interfaces. The RGD motif of fibronectin binds αvβ3and α5β1 integrins to promote neuritogenesis by PC12 cells [16, 17]. Whereas, interaction of integrin α8β1 with RGD sequence in cell adhesion molecule stimulates neurite outgrowth from dorsal root ganglion . In PC12 cells, RGD-mediated adhesion triggers many signaling events including focal adhesion kinase phosphorylation and cytoskeletal reorganization [17, 18]. Whether these signaling cascades occur in response to [RGD-V6
20 remains to be studied.
The spontaneous increase in Tuj1 expression under non-differentiating conditions was in contrast to the effect of the poly(RGD-VPGIG) peptide, which supports neurite extension from PC-12 cells when nerve growth factor is present in the growth media . However, up-regulation of the neuronal markers TuJ1, MAP2, and GFAP in the cells grown on [RGD-V6
20 confirmed that [RGD-V6
20 is effective in stimulating neuronal cell differentiation. Moreover, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated in the presence of both [RGD-V6
20 and RA. This additive effect indicates that [RGD-V6
20 enhances the differentiation response of neuronal cells to RA. This is important for eventual in vivo application of matrix protein in combination with differentiation-stimulating agents.