Functionalized α-Helical Peptide Hydrogels for Neural Tissue Engineering

Abstract

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Keywords: RGD peptide; hydrogel; nerve tissue engineering; peptide; self-assembly; stem cell.

Figure 1

Nestin expression and proliferation of NSCs on hSAF and laminin gels. (A–D) Single-cell suspension of GFP-nestin positive murine NSCs (green) seeded on (A) laminin, (B) undecorated hSAF, and (C) RGDS-decorated hSAF gels. Main panels, Day 14; insets, Day 0. (A–C) Day 14. (D) Proliferative activity of NSCs on laminin (red), undecorated hSAF (blue), and RGDS-decorated hSAF (green) gels over 14 days as measured by an MTT assay (n = 3 for each time point).

Figure 2

3D neurosphere formation by NSCs in hSAF gels. 3D reconstruction of z-stack fluorescent images on (A) laminin, (B) undecorated hSAF, and (C) RGDS-decorated hSAF gels. (D) 3D reconstruction of z-stack fluorescent images of cells on RGDS-decorated gels showing neurosphere connections. DAPI-stained cell nuclei (blue) and nestin expression (green). (A–D) Grid scales: 24.75 μm.

Figure 3

Migration of NSCs across half-moon hSAF, and laminin gels. (A–H) First and last frames of NSC migration captured over 24 h. (A) Bright-field images of NSCs on laminin, (B) undecorated hSAF, (C) RGDS-decorated hSAF gels, and (D) the border between an undecorated and RGDS-decorated hSAF half-moon gel. Blue arrowheads indicate the direction of cell migration. (E–H) Final frame bright-field images of NSCs on (E) laminin, (F) undecorated hSAF, (G) RGDS-decorated hSAF gels, and (H) the border between an undecorated and RGDS-decorated hSAF half-moon gel at 24 h postseeding. Migration tracks (red) indicate the overall movement over this period; and the arrowheads indicate direction of cell migration. (I–K) Migration tracks of cells over 24 h on (I) laminin, (J) undecorated hSAF, (K) RGDS-decorated hSAF gels, and (L) the border between an undecorated and RGDS-decorated hSAF half-moon gel represented as spider plots. Each line indicates a separate cell showing the start (center of the plot) and end (black dot) positions of each cell. (M) Displacement of cells over 24 h. (N) Directedness of cells over 24 h: migration in the y-direction (cos θ = 0), to the right (cos θ = +1), and to the left (cos θ = −1) are presented. n = 15 (laminin), 24 (undecorated hSAF), 48 (RGDS-decorated hSAF), and 12 (border between undecorated and RGDS-decorated gels).

Figure 4

Differentiation of NSCs on hSAF and laminin gels. (A, D, G) laminin; (B, E, F) undecorated hSAF; (C, F, I) RGDS-decorated hSAF gels. (A–C) Phase-contrast images showing cell morphology at day 7. (D–F) Representative fluorescent images show NSC expression of MAP2 (red) and DAPI-stained nuclei (blue) on all gels. (G–I) Representative fluorescent images show NSC expression of GFAP (red) and DAPI-stained nuclei (blue) on all gels. (J) Percentage of cells producing processes after 14 days in culture on laminin (red), undecorated hSAF (blue) and RGDS-decorated hSAF (green) gels. Percentage of cells expressing the neuronal differentiation markers (K) MAP2 and (L) GFAP. n = 3 for all experiments.

Figure 5

Activation dynamics of NSC K⁺ currents on RGDS-decorated and undecorated hSAF gels at (A, C) 7 and (B, D) 14 days. (A, B) Outward K⁺ currents evoked by a +80 mV voltage step, normalized to the plateau and averaged on undecorated hSAF (black) and RGDS-decorated hSAF gel (red). (C, D) Analysis of the specific conductance recorded on undecorated hSAF (black) and RGDS-decorated hSAF gel (red) with different voltage step intensities. The Boltzmann sigmoidal fit of the conductance-voltage curves at each time-point and for each gel is shown. The extrapolated maximal conductance and relative values for half the voltage that generates half the maximal conductance are reported. n = 12 (undecorated hSAF) and 14 (RGDS-decorated hSAF) for day 7 and 5 (undecorated hSAF) and 10 (RGDS-decorated hSAF) for day 14.