Synthesis and Application of Scaffolds of Chitosan-Graphene Oxide by the Freeze-Drying Method for Tissue Regeneration

Abstract

Several biomaterials, including natural polymers, are used to increase cellular interactions as an effective way to treat bone injuries. Chitosan (CS) is one of the most studied biocompatible natural polymers. Graphene oxide (GO) is a carbon-based nanomaterial capable of imparting desired properties to the scaffolds. In the present study, CS and GO were used for scaffold preparation. CS was extracted from the mycelium of the fungus Aspergillus niger. On the other hand, GO was synthesized using an improved Hummers-Offemann method and was characterized by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), and dynamic light scattering (DLS). Subsequently, three formulations (GO 0%, 0.5%, and 1%) were used to prepare the scaffolds by the freeze-drying technique. The scaffolds were characterized by FTIR, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM), to determine their thermal stability and pore size, demonstrating that their stability increased with the increase of GO amount. Finally, the scaffolds were implanted, recollected 30 days later, and studied with an optical microscope, which evidenced the recovery of the tissue architecture and excellent biocompatibility. Hence, these results strongly suggested the inherent nature of chitosan/graphene oxide (CS/GO) scaffolds for their application in bone tissue regeneration.

Keywords: chitosan; freeze-drying method; graphene oxide; scaffolds.

Figure 1

Fourier transform infrared spectroscopy (FTIR) spectrum of the graphene oxide (GO) synthesized in the study.

Figure 2

X-ray diffractogram of the GO synthesized in our study.

Figure 3

(a) Atomic force microscopy of sectioned GO plates (b) sectional thickness of each plate according to its height.

Figure 3

(a) Atomic force microscopy of sectioned GO plates (b) sectional thickness of each plate according to its height.

Figure 4

Pictures of the freeze-dried scaffolds of chitosan (CS) (a) without graphene oxide (GO), (b) with 0.5% of GO and (c) with 1.0% of GO.

Figure 5

Fourier transform infrared spectroscopy (FTIR) spectrum of the chitosan/graphene oxide (CS/GO) scaffolds synthesized.

Figure 6

Scanning electron microscopy of chitosan (CS) scaffolds (a) without graphene oxide (GO), (b) with 0.5% GO and (c) with 1.0% GO.

Figure 7

Image (A) shows the dorsal area where the surgical preparation was made; image (B) shows the internal surface of the skin, the three samples encapsulated by a scar tissue are appreciated. 1: chitosan/graphene oxide (CS/GO) 0%; 2: CS/GO 0.5% and 3: CS/GO 1%.

Figure 8

Rat skin sample implanted with collagen film. 1: Epidermis, 2: Dermis, 3: Subcutaneous cellular tissue. Three corresponds to the implantation area, BV: Blood vessel. Hematoxylin-Eosin Technique. Image (A) is at 4× magnification, Image (B) at 10×, and Image (C) at 40×.

Figure 9

Chitosan/graphene oxide (CS/GO) 0% films implanted in rat skin; in the pictures (A,B), movies are seen at 4× and 10×, respectively, using the Hematoxylin-Eosin Technique; in the images (C,D), at 40×, the films in the process of and surrounded by a mixed inflammatory infiltrate are observed, the D image is realized using Masson’s Trichromacy technique. 1: epidermis, 2: Dermis. 3: subcutaneous cellular tissue (implantation zone), 4. Mixed inflammatory infiltrates, 5. Scaffolds of CS/GO 0%.

Figure 10

Scaffolds of chitosan/graphene oxide (CS/GO) 0.5% implanted in rat skin; in the images, (A,B) scaffolds are appraised to 4× and 10×, respectively, using the Hematoxylin-Eosin Technique. Images (C,D), at 40×, are scaffolds in the process of degradation and are surrounded by a mixed inflammatory infiltrate. Image (D) is made using Masson’s trichromacy technique; in this image we can see a fibrous tissue capsule surrounding the CS/GO scaffolds. 1: epidermis, 2: Dermis. 3: subcutaneous cellular tissue (implantation zone), 4. Mixed inflammatory infiltrates, 5. Scaffolding CS/GO 0.5%, 6: Fibrous capsule. Technique. Image (A) is at 4× magnification, Image B at 10×. Image (C) at 40×.

Figure 11

Scaffolds of CS/GO 1% implanted in rat skin. In the images (A,B), the scaffolds are appreciated at 4× and 10×, respectively, using the Hematoxylin-Eosin Technique. In the image (C) at 40×, the film is observed in the process of resorption and surrounded by a mixed inflammatory infiltrate. The image (D) at 10× is made using Masson’s trichromacy technique. In this image, a fibrous tissue capsule is seen surrounding the scaffold of CS/GO. 1: Epidermis, 2: Dermis. 3: subcutaneous cellular tissue (implantation zone), 4. Mixed inflammatory infiltrates, 5. CS/GO 1%, 6: Fibrous capsule.