Induced Nucleation of Biomimetic Nanoapatites on Exfoliated Graphene Biomolecule Flakes by Vapor Diffusion in Microdroplets

Abstract

The nucleation of apatite nanoparticles on exfoliated graphene nanoflakes has been successfully carried out by the sitting drop vapor diffusion method, with the aim of producing cytocompatible hybrid nanocomposites of both components. The graphene flakes were prepared by the sonication-assisted, liquid-phase exfoliation technique, using the following biomolecules as dispersing surfactants: lysozyme, L-tryptophan, N-acetyl-D-glucosamine, and chitosan. Results from mineralogical, spectroscopic, and microscopic characterization (X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman, Variable pressure scanning electron microscopy (VPSEM), and transmission electron microscopy (TEM)) indicate that flakes were stacked in multilayers (> 5 layers) and most likely intercalated and functionalized with the biomolecules, while the apatite nanoparticles were found forming a coating on the graphene surfaces. It is worthwhile to mention that when using chitosan-exfoliated graphene, the composites were more homogeneous than when using the other biomolecule graphene flakes, suggesting that this polysaccharide, extremely rich in –OH groups, must be arranged on the graphene surface with the –OH groups pointing toward the solution, forming a more regular pattern for apatite nucleation. The findings by XRD and morphological analysis point to the role of “functionalized graphene” as a template, which induces heterogeneous nucleation and favors the growth of apatite on the flakes’ surfaces. The cytocompatibility tests of the resulting composites, evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose–dependent manner on GTL-16 cells, a human gastric carcinoma cell line, and on m17.ASC cells, a murine mesenchymal stem cell line with osteogenic potential, reveal that in all cases, full cytocompatibility was found.

Vapour Diffusion Sitting Drop Method to Induce Nucleation of Calcium Phosphate on Exfoliated Graphene and Graphene Oxide Flakes

“…Our laboratory has developed a biomimetic route, based on the vapour diffusion sitting drop micro-method (hereafter SDVD) [13], which has been used to induce the heterogeneous nucleation and growth of CaP films on mica sheets [14] and exfoliated multilayer graphene (G) [15]. The obtained nAps by this method are Ca-and OH-deficient and, in addition, are doped with CO 3 2− ions.…”

“…The LPE technique produced multilayer flakes. The SDVD method is performed in a closed environmental microreactor called crystallization mushroom, which is composed of a crystallization chamber in the upper part, and a gas generation chamber on the lowest side containing NH 4 HCO 3 [13][14][15]. We deposit 12 aqueous Ca 2+ -and HPO 4 2− -bearing droplets arranged circularly in the crystallization chamber.…”

Biological Crystallization

“…Due to their excellent biocompatibility, osteoconductivity and osteoinductivity, these nanoparticles find application in the field of biomaterials. In the fifth paper, Gómez Morales et al [5] explore the nucleation of apatite nanoparticles on exfoliated graphene flakes to yield graphene/apatite nanocomposites with applications as bone grafts.…”