Assessing cellular response to functionalized α-helical peptide hydrogels

Abstract

α-Helical peptide hydrogels are decorated with a cell-binding peptide motif (RGDS), which is shown to promote adhesion, proliferation, and differentiation of PC12 cells. Gel structure and integrity are maintained after functionalization. This opens possibilities for the bottom-up design and engineering of complex functional scaffolds for 2D and 3D cell cultures.

Keywords: 2D cell culture; PC12 cells; peptide hydrogels; self-assembled fibers; tissue engineering.

Figure 1

Peptide sequences, a schematic of the click reaction, and the half-moon model. A) Peptide sequences used for this study. Key: z, azido norleucine; Prop, propiolate. B) The gel was formed using an N-terminally azido-modified hSAF-p1. Decoration was achieved by performing a click reaction with alk-RGDS on azide-containing gels catalyzed by CuSO₄ with ascorbic acid (AA). C) Side-by-side gel formation in 24-well cell-culture plates allowed a direct comparison of cellular behavior on undecorated hSAF- and RGDS-decorated hSAF gels. Key: undecorated hSAF gel, gray; and RGDS-decorated hSAF gel, blue.

Figure 2

Fiber morphology and gel structure. Transmission electron images for the A) undecorated hSAF and C) RGDS-decorated hSAF fibers. Average fiber diameters were 13 ± 5 nm for hSAF-undecorated fibers and 17 ± 4 nm for RGDS-decorated hSAF fibers. B,D) Scanning electron images showing interconnected fibers forming porous hydrogels of similar morphology D) with and B) without alk-RGDS. The gels are self-supporting (insets). Scale bars on (A,C) equal 200 nm while scale bars on (B,D) equal 1 μm.

Figure 3

Response of PC12 cells to hydrogels. A,D) Light microscopy images showing PC12 attachment, and elongated cell morphology, to undecorated hSAF- and RGDS-decorated hSAF gels after 14 d. B,E) Representative fluorescent images for DAPI-stained cells on undecorated hSAF- and RGDS-decorated hSAF gels. C,F) Viable cells on undecorated hSAF- and RGDS-decorated hSAF gels indicated by calcein-AM staining. G) Proliferation of PC12 cells on gels and TCP over 14 d as judged by MTT assays. H) DNA quantification using Hoechst dye for PC12 cells on the gels and TCP over 14 d. I) PC12 differentiation, J) number of neurite-like processes, and K) lengths of processes as a function of time. Due to a high proliferation rate, individual cell processes were difficult to identify at day 14 on Matrigel. Dashed lines represent the projections for Matrigel assuming that the underlying trend from the early time points continues. Key: undecorated hSAF gel, gray; RGDS-decorated hSAF gel, blue; Matrigel, red; and TCP, green.