Synthesis and fabrication of films including graphene oxide functionalized with chitosan for regenerative medicine applications

Abstract

Graphene oxide (GO) has recently gained attention as a scaffold reinforcing agent for tissue engineering. Biomechanical and biological properties through a synergistic effect can be strengthened when combined with other materials such as chitosan (CS). For that reason, chitosan was used for Graphene Oxide (GO) functionalization through an amide group whose formation was evident by bands around 1600 cm^-1 in the FTIR analysis. Furthermore, bands located at 1348 cm^-1 (D band), 1593 cm^-1 (G band), and 2416 cm^-1 (2D band) in the RAMAN spectrum, and the displacement of the signal at 87.03 ppm (C5) in solid-state ¹³C-NMR confirmed the amide formation. Films including the CS-GO compound were prepared and characterized by thermogravimetric analysis (TGA), where CS-GO film presented a lighter mass loss (~10% less loosed) than CS due probably to the covalent functionalization with GO, providing film thermal resistance. The CS-GO films synthesized were implanted in Wistar rats' subdermal tissue as a first approximation to the biological response. In vivo tests showed a low inflammatory response, good cicatrization, and advanced resorption at 60 days of implantation, as indicated by histological images. It was evidenced that the covalent union between CS and GO increased biocompatibility and the degradation/resorption capacity, demonstrating tissue regeneration with typical characteristics and tiny remnants of implanted material surrounded by a type III collagen capsule. These results show the potential application of the new synthesized films, including the CS-GO compound, in tissue engineering.

Keywords: Chitosan; Graphene oxide; Regenerative medicine; Tissue engineering.

Figure 1

Raman characterization of a) CS-GO and b) GO compounds.

Figure 2

Fourier transform infrared spectroscopy (FTIR) characterization of a) GO, b) CS, and c) CS-GO.

Figure 3

The solid-state ¹³C-NMR characterization of CS.

Figure 4

The solid-state ¹³C-NMR characterization of CS-GO.

Figure 5

Thermogravimetric analysis of a) CS, b) CS-GO, and c) GO.

Figure 6

Macroscopic appearance of the implanted area in Wistar rat after 30 days of implantation. A. Macroscopically appearance of the skin. B. External surface of the skin. C. Internal surface of the skin. IZ: Implantation Zone. CS-GO: Chitosan-Graphene Oxide membrane.

Figure 7

Microscopic images at 4× of CS-GO: Chitosan-Graphene Oxide. A. Hematoxylin-Eosin Technique. B. Gomori trichrome technique. IZ: Implantation Zone. E: Epidermis. D: Dermis. H: Hypodermis. M: Muscle.

Figure 8

Gomori's trichrome technique images of CS-GO film implanted at 30 days. A. At 40×. B. At 100×. BV: Blood vessels. FC: Fibrous capsule. CS-GO: Chitosan-Graphene Oxide. RB: Red blood cells. Mf: Macrophages. H: Histiocytes.

Figure 9

Masson's trichrome technique images at 10× of CS and CS-GO films implanted at 30 days. A. CS films B. CS-GO films. IZ: Implantation Zone. FC: Fibrous capsule. CS-GO: CS-GO membrane. CS: Chitosan film.

Figure 10

Masson's trichrome technique images at 4× of CS-GO and CS/GO films implanted at 60 days. A. CS-GO films. B. CS/GO films. IZ: Implantation zone. FC: Fibrous capsule. CS-GO: Chitosan-Graphene Oxide. CS/GO: Chitosan/Graphene Oxide physical mixture.

Figure 11

Masson's trichrome technique images at 10× of CS-GO and CS/GO films implanted at 60 days. A. CS-GO films. B. CS/GO films. FC: Fibrous Capsule. CS-GO: Functionalized Chitosan-Graphene Oxide. CS/GO: Chitosan/Graphene Oxide physical mixture. Stars: CS-GO fragments in the process of degradation/reabsorption.

Figure 12

Masson's Trichrome Technique images at 40× of CS-GO and CS/GO films implanted at 60 days. A. CS-GO films. B. CS/GO films. BV: Blood vessel. CS-GO: Chitosan-Graphene Oxide covalently functionalized. CS/GO: Chitosan/Graphene Oxide Physical Mixture.