Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures

Abstract

The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering. We investigated the self-assembled nanostructures of functionalized peptides featuring four, two or no glycine-spacers between the self-assembly sequence RADA16-I and the functional biological motif PFSSTKT. The effectiveness of their biological functionalization was assessed via in vitro experiments with neural stem cells (NSCs) and their molecular assembly was elucidated via atomic force microscopy, Raman and Fourier Transform Infrared spectroscopy. We demonstrated that glycine-spacers play a crucial role in the scaffold stability and in the exposure of the functional motifs. In particular, a glycine-spacer of four residues leads to a more stable nanostructure and to an improved exposure of the functional motif. Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation. Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

Keywords: AFM; FTIR; Micro-Raman; biomaterial; nanostructure; neural stem cell.

Figure 1

The functionalized self-assembling peptides used in this study. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1. A spacer of two (B) or four glycines (C) (colored in green) was inserted between the self-assembling cores and the functional motif PFSSTKT.

Figure 2

AFM images of FPs solutions at a concentration of 0.05% (w/v). 2G-BMHP1 (B) and 4G-BMHP1 (C) self-assemble into nanofibers (average width: ∼15 nm). 0G-BMHP1 (A) self-organizes into nanofibers shorter than (B) and (C) but featuring similar width. Inserts show high-resolution images of single or few fibers of each peptide at a concentration of 0.01% (w/v).

Figure 3

ATR/FTIR absorption spectra of self-assembling peptides. (A) ATR/FTIR absorption spectra of self-assembling peptides. (B) Second derivatives of ATR/FTIR spectra in different buffer conditions. Spectra show the two intermolecular β-sheet bands around 1618 and 1696 cm⁻¹. The highest intensity of the β-sheet components is observed for 4G-BMHP1 in assembling conditions of buffer 3 ((B), buffer 3). 0G-BMHP1 displays an up shift of the 1618 cm⁻¹ component in buffer 4 ((B), buffer 4), indicating a loosely packed assembly.

Figure 4

Raman Spectra of tested self-assembling peptides. (A) Raman Spectra (1600–1800 cm⁻¹ region) of the tested self-assembling peptides. Each spectrum shows the peptide Amide I region in all buffered solutions. In buffer 1, buffer 2 and buffer 3, each peptide shows a similar conformation of Amide I peak, suggesting a similar β-sheet conformation of the peptide centered in 1675 cm⁻¹. In buffer 4 the pH shift alters the self-assembled structure of 0G-BMHP1 thus affecting the shape of its Amide I peak and suggesting a wider unordered component. (B) Raman Spectra of 2500–3100 cm⁻¹ region of the tested self-assembling peptides (region of νCH₂) exposed to different buffers. The change in the relative intensity between the peak at ∼2940 cm⁻¹ (νCH₂) and the peak at ∼3061 cm⁻¹ νCH₂ of Phe can be appreciated in case of peptide 4G BMHP1.

Figure 5

Thermal stability of the self-assembled peptides. Temperature dependence of the intermolecular β-sheet band intensity at 1618 cm⁻¹ of the self-assembly peptides in D₂O solution, from 30°C to 100°C. The FPs show a higher stability in comparison with RADA16-I. Noteworthy, 0G-BMHP1 at 100°C reduces its 1618 cm⁻¹ peak intensity at ∼65% similarly to RADA16-I, while 2G-BMHP1 and 4G-BMHP1 at the same temperature decrease to ∼74%. Standard deviation of the data from independent experiments is smaller than the symbol size.

Figure 6

Phase contrast images of differentiating neural stem cells (7 days in vitro) over the tested scaffolds. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1 SAPeptides. (D) Positive and (E) negative controls. 4G-BMHP1 coaxes NSCs to differentiate and survive most effectively while, in case of 0G-BMHP1, small clusters of poorly adhered NSCs testify a possible poor availability of the BMHP1 functional motif for cell membrane receptor binding and consequently cell differentiation pathways activation. Additionally, the “sinking” of NSCs within the assembled scaffold of 0G-BMHP1 (A) testifies a possible lower mechanical stiffness insufficient to bear the weight of NSCs seeded over the top surface of the substrate. Scale bars are 100 μm. CellTiter results (F) show significant differences for all possible coupled experimental groups (P < 0.05) except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean.

Figure 7

Quantitative NSCs differentiation assay. Data are expressed as percentages of cells positive for GFAP, β-Tubulin, GALC/O4 and Nestin markers over the total number of cells counted in the same fields (see Materials and Methods for details). NSCs have been differentiated for 7 days in vitro. By increasing the length of the Gly-spacer, the percentage of Nestin⁺ significantly decreased. Despite that GFAP⁺ cell fraction increased inversely. β-Tubulin⁺ and GalC/O4⁺ cell percentages did not significantly differ among the tested substrates and assembled SAPs. NSCs cultured over plastic well surfaces (Negative Control), being composed of clusters of mainly Nestin⁺ cells, were not included in this graph. Values are expressed as means ± standard error of the mean. * = significantly different values (P < 0.05).