Graphene oxide containing self-assembling peptide hybrid hydrogels as a potential 3D injectable cell delivery platform for intervertebral disc repair applications

Abstract

Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP). In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. STATEMENT OF SIGNIFICANCE: Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells' microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.

Keywords: Cell culture; Cell delivery; Graphene oxide; Hydrogels; Injectable; Nucleus pulposus; Peptide; Tissue engineering.

Graphical abstract

Fig. 1

Top: Chemical structure of FEFKFEFK peptide (A), SEM image of GO flakes (Bi) and flake size distribution (Bii). Bottom: Schematic representation of the formulation route used to prepare peptide/GO hybrid hydrogels (C).

Fig. 2

Top: Inverted vials photographs of peptide hydrogels (A) and peptide-GO hybrid hydrogels (B) taken one hour after formulation. Bottom: Light microscopy images of GO-F810 (C), GO-F815 (D) and GO-F820 (E) hydrogels (Inset: photographs of corresponding hydrogel droplets, 30 µL each – Scale bar 2 mm).

Fig. 3

Top: FTIR spectra of peptide (A) and peptide-GO hybrid (B) hydrogels. C) TEM images of hydrogels formulated without (top row) and with (bottom row) GO. (Asterisks highlight GO flakes embedded in the peptide fibrillar matrix).

Fig. 4

Top: AFM images of GO flakes embedded in the peptide matrix at 5 µm × 5 µm (A) and 0.7 µm × 0.7 µm (B) scan sizes. Bottom: a) to c) height profiles corresponding to the scans shown in (B) and d) schematic model of β-sheet fibres and β-sheet fibrils templated on GO surface.

Fig. 5

Storage moduli at 1 Hz of peptide hydrogels as formulated at pH = 4 (A) and after overnight media conditioning at pH = 7 (B). All measurements were performed at least 3 times at 37 °C. (Data are shown as mean ± SD; ^***p-value <0.001; ^**p-value <0.05).

Fig. 6

Left: Shear-thinning and recovery experiments demonstrating hydrogels’ injectability (Top: F820 formulated without GO; Bottom: GO-F820 formulated with GO). Right: Photographs of hydrogels being injected in a Petri dish (C & E: Hydrogel formulated without GO; D & F: Hydrogels formulated with GO) and in PBS (G: hydrogel formulated without GO; H: Hydrogel formulated with GO) (Pink dye was added to the hydrogels formulated without GO to enhance contrast). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

LIVE/DEAD assay (green = calcein AM for viable cells and red = ethidium homodimer-1 for dead cells) was used to assess cell viability of BNPCs. Viability was evaluated at day 1, 4 and 7 after encapsulation. Viable cells are show green fluorescence, dead cells show red fluorescence. Scale bar = 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Percentage of viable cells extracted from LIVE/DEAD images for A) peptide hydrogels and B) hybrid hydrogels. Metabolic activity for cells encapsulated in C) peptide hydrogels and D) hybrid hydrogels. Data are shown as mean ± SD (^***p-value <0.001; ^**p-value <0.05).

Fig. 9

Size frequency plots of cell clusters counts obtained using LIVE/DEAD assay. A, C, E) Size frequency plots of cell clusters encapsulated in peptide hydrogels without GO, i.e. F810, F815 and F820 respectively. B, D, F) Size frequency plots of cell clusters encapsulated in peptide hydrogels with GO, i.e. GO-F810, GO-F815 and GO-F820 respectively. D50: Cell/cell cluster area value below which 50% of the cells/cell clusters area distribution is contained. Red arrows indicate shift for D50 values at day 7 compared to day 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)