Reduced graphene oxide-loaded nanocomposite scaffolds for enhancing angiogenesis in tissue engineering applications

Abstract

Tissue engineering combines cells, scaffolds and signalling molecules to synthesize tissues in vitro. However, the lack of a functioning vascular network severely limits the effective size of a tissue-engineered construct. In this work, we have assessed the potential of reduced graphene oxide (rGO), a non-protein pro-angiogenic moiety, for enhancing angiogenesis in tissue engineering applications. Polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) scaffolds loaded with different concentrations of rGO nanoparticles were synthesized via lyophilization. Characterization of these scaffolds showed that the rGO-loaded scaffolds retained the thermal and physical properties (swelling, porosity and in vitro biodegradation) of pure PVA/CMC scaffolds. In vitro cytotoxicity studies, using three different cell lines, confirmed that the scaffolds are biocompatible. The scaffolds containing 0.005 and 0.0075% rGO enhanced the proliferation of endothelial cells (EA.hy926) in vitro. In vivo studies using the chick chorioallantoic membrane model showed that the presence of rGO in the PVA/CMC scaffolds significantly enhanced angiogenesis and arteriogenesis.

Keywords: angiogenesis; nanocomposite scaffolds; reduced graphene oxide; tissue engineering; vascularization.

Figure 1.

(a) FT-Raman spectra of GO and rGO, (b) TEM micrograph and (c) particle-size distribution of rGO.

Figure 2.

(a) FT-Raman spectra and (b) XRD patterns of PVA/CMC, PVA/CMC rGO 0.0025%, PVA/CMC rGO 0.005%, PVA/CMC rGO 0.0075% and PVA/CMC rGO 0.01%.

Figure 3.

SEM micrographs (a) PVA/CMC, (b) PVA/CMC rGO 0.0025%, (c) PVA/CMC rGO 0.005%, (d) PVA/CMC rGO 0.0075%, (e) PVA/CMC rGO 0.01% and (f) porosity values of the scaffolds.

Figure 4.

(a) DSC and (b) TGA thermograms of PVA/CMC, PVA/CMC rGO 0.0025%, PVA/CMC rGO 0.005%, PVA/CMC rGO 0.0075% and PVA/CMC rGO 0.01%.

Figure 5.

(a) Swelling behaviour and (b) in vitro biodegradation of PVA/CMC, PVA/CMC rGO 0.0025%, PVA/CMC rGO 0.005%, PVA/CMC rGO 0.0075% and PVA/CMC rGO 0.01%.

Figure 6.

(a) Cell viability of NIH3T3, ECV304 and EA.hy926 cultured on the scaffolds, (b) phase-contrast (column one), DAPI-stained (column two), FDA-stained (column three) and SEM micrographs (column four) of EA.hy926 cultured on the scaffolds and (c) cell proliferation of EA.hy926 cultured on scaffolds up to for 72 h (n = 6, **p < 0.01 versus control).

Figure 7.

(a) Digital images of the untreated and treated CAM and (b) percentage increase in the average number of blood vessels and average thickness of blood vessels obtained on day 10 of the CAM assay. The values are normalized to that of the untreated control on day 8 (n = 9, **p < 0.01 and ***p < 0.001 versus control).