Reduced Graphene Oxide-Extracellular Matrix Scaffolds as a Multifunctional and Highly Biocompatible Nanocomposite for Wound Healing: Insights into Characterization and Electroconductive Potential

Abstract

The development of novel regenerative technologies based on the implementation of natural extracellular matrix (ECM), or individual components of ECM combined with multifunctional nanomaterials such as graphene oxide and reduced graphene oxide, has demonstrated remarkable results in wound healing and tissue engineering. However, the synthesis of these nanocomposites involves great challenges related to maintaining the biocompatibility with a simultaneous improvement in their functionalities. Based on that, in this research we developed novel nanoengineered ECM-scaffolds formed by mixing small intestinal submucosa (SIS) with graphene oxide (GO)/reduced graphene oxide (rGO) to improve electrical conductivity while maintaining remarkable biocompatibility. For this, decellularized SIS was combined with GO to form the scaffold precursor for subsequent lyophilization, chemically crosslinking and in situ reduction. The obtained GO and rGO were characterized via Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), electrical conductivity testing and atomic force microscopy (AFM). The results confirm the suitable synthesis of GO, the effective reduction to rGO and the significant increase in the electrical conductivity (more than four orders of magnitude higher than bare GO). In addition, the graphene oxide/reduced graphene oxide-SIS scaffolds were characterized via Raman spectroscopy, FTIR, TGA, SEM, porosity assay (higher than 97.5% in all cases) and protein secondary structural analysis. Moreover, the biocompatibility of scaffolds was studied by standardized assays of hemolysis activity (less than 0.5%), platelet activation and deposition, and cell viability in Vero, HaCat and HFF-1 cells (higher than 90% for all evaluated cell lines on the different scaffolds). The obtained results confirm the remarkable biocompatibility, as supported by high hemocompatibility, low cytotoxicity and no negative impact on platelet activation and deposition. Finally, structural characteristics such as pore size and interconnectivity as well as superior cell attachment abilities also corroborated the potential of the developed nanoengineered ECM-scaffolds as a multifunctional nanoplatform for application in regenerative medicine and tissue engineering.

Keywords: conductive scaffolds; electrostimulation therapy; extracellular matrix; graphene oxide; reduced graphene oxide; regenerative medicine; tissue engineering; wound healing.

Figure 1

Characterization of GO and rGO. (A) Schematic representation of the chemical structure of GO and rGO. (B) GO/ultra−pure ethanol solution (1) and rGO/ultra−pure ethanol solution (2). (C) Raman spectra (514 nm) of graphite, GO and rGO. (D) FTIR spectra of graphite, GO and rGO. (E) TGA thermograms for graphite, GO and rGO. (F) XRD pattern of GO. (G) Electrical conductivity of GO and rGO films. (H) GO and rGO films (deposited on glass slides).

Figure 2

(A) AFM image of GO. (B,C) correspond to more detailed observations of GO nanosheets in Figure 2A. The height profiles of (B,C) GO nanosheets are shown in (D,E), respectively.

Figure 3

Characterization of the scaffolds. (A) Raman spectra of SIS-rGO scaffold. (B) TGA and (C) FTIR analysis of the developed nanocomposites. (D) Second derivative of the FTIR spectra in range of the amide I band (1700−1600 cm⁻¹).

Figure 4

SEM images of hydrated and completely dry scaffolds. Pores appear homogenously distributed and well interconnected.

Figure 5

Porosity of the scaffolds calculated by the liquid displacement method. Results with a p-value ≤ 0.05 (*) were considered statistically different. Symbol * corresponds to statistically significant difference with a p-value in the range of 0.01 ≤ p-value ≤ 0.05, ** to statistically significant difference with a p-value in the range of 0.001 ≤ p-value < 0.01, *** to p-value in the range of 0.0001 ≤ p-value ≤ 0.001 and **** to p-value < 0.0001.

Figure 6

Hemolytic effect of the nanocomposites tested by extracts (A) and direct contact (B). (C) Cell viability of the different scaffolds in Vero, HaCat and HFF-1 cells (Alamar blue assay). Platelet activation tested by LDH quantification assay (D) and SEM images of platelet deposition (E) evaluated in crosslinked SIS, SIS-GO and SIS-rGO scaffolds. Red arrows show activated platelets. Results with a p-value ≤ 0.05 (*) were considered statistically different. Symbol * corresponds to statistically significant difference with a p-value in the range of 0.01 ≤ p-value ≤ 0.05, ** to statistically significant difference with a p-value in the range of 0.001 ≤ p-value < 0.01, *** to p-value in the range of 0.0001 ≤ p-value ≤ 0.001 and **** to p-value < 0.0001.

Figure 7

(A) Analysis of Vero cell morphology via confocal imaging. Yellow arrows present cells that exposed the typical elongated morphology confirming strong adhesion to the matrix, and white arrows expose cells with irregular round-shape morphology indicating poor adhesion to the matrix. (B) Three-dimensional reconstruction of cell distribution of Vero cells in all the developed scaffolds (confocal images 20×). Cell nuclei were stained with Hoechst 33342 (Blue) and actin filaments with Alexa Fluor 594 Phalloidin (Red).