Antimicrobial and Wound-Healing Activities of Graphene-Reinforced Electrospun Chitosan/Gelatin Nanofibrous Nanocomposite Scaffolds

Abstract

This study aims at preparing electrospun chitosan/gelatin nanofiber scaffolds reinforced with different amounts of graphene nanosheets to be used as antibacterial and wound-healing scaffolds. Full characterization was carried out for the different fabricated scaffolds before being assessed for their antimicrobial activity against Escherichia coli and Staphylococcus aureus, cytotoxicity, and cell migration capacity. Raman and transmission electron microscopies confirmed the successful reinforcement of nanofibers with graphene nanosheets. Scanning electron microscopy and porosity revealed that nanofibers reinforced with 0.15% graphene nanosheets produced the least diameter (106 ± 30 nm) and the highest porosity (90%), in addition to their good biodegradability and swellability. However, the excessive increase in graphene nanosheet amount produced beaded nanofibers with decreased porosity, swellability, and biodegradability. Interestingly, nanofibers reinforced with 0.15% graphene nanosheets showed E. coli and S. aureus growth inhibition percents of 50 and 80%, respectively. The cell viability assay showed no cytotoxicity on human fibroblasts when cultured with either unreinforced or reinforced nanofibers. The cell migration was higher in the case of reinforced nanofibers when compared to the unreinforced nanofibers after 24 and 48 h, which is substantially associated with the great effect of the graphene nanosheets on the cell migration capability. Unreinforced and reinforced nanofibers showed cell migration results up to 93.69 and 97%, respectively, after 48 h.

Scheme 1. Schematic Diagram Illustrating the Fabrication Procedure of Nanofiber Scaffolds

Figure 1

SEM images of (a) CS/GL film, (b) CS/GL NFs, (c) 0.10% GNS-CS/GL NFs, (d) 0.15% GNS-CS/GL NFs, and (e) 0.20% GNS-CS/GL NFs and their corresponding histograms. The yellow arrows point to the formed beads.

Figure 2

SEM images of cross-linked (a) CS/GL NFs, (b) 0.10% GNS-CS/GL NFs, (c) 0.15% GNS-CS/GL NFs, and (d) 0.20% GNS-CS/GL NFs, and (e) TEM image of 0.15% GNS-CS/GL NFs (yellow arrows point at the presence of GNS within the nanofibers).

Figure 3

FTIR spectra of CS (top, light gray), GL (middle, medium gray), and CS/GL (bottom, dark gray) NFs.

Figure 4

Raman spectra of 0.15% GNS-CS/GL NFs (top) and CS/GL NFs (bottom). The arrows point to the three characteristic bands of GNS.

Figure 5

Porosity % of each of the fabricated nanofibers scaffolds (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Figure 6

(a) Swelling profile of the different fabricated scaffolds along 3 h and (b) swelling maximum after 2 h (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Figure 7

(a) Biodegradability of the different fabricated scaffolds over 21 days and (b) weight remaining percentage after the first, second, and third weeks (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Figure 8

(a) E.coli growth and CFU enumeration, (b) S. aureus growth and CFU, (c) E.coli growth inhibition percent, and (d) S. aureus growth inhibition percent. (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).

Figure 9

(a) Cell viability of both nanofibers, and cell adhesion on (b) CS/GL NFs and (c) GNS-CS/GL NFs.

Figure 10

In vitro wound-healing assay against the negative control, CS/GL NFs, and GNS-CS/GL NFs at 0, 24, and 48 h.

Figure 11

Percent of in vitro wound closure against normal human fibroblast: (a) after 24 h and (b) after 48 h.