Graphene–Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo

Abstract

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Graphene Oxide-Modified Concentric Microgrooved Titanium Surfaces for the Dual Effects of Osteogenesis and Antiosteoclastogenesis

“…In addition to improving cell proliferation, many studies have confirmed that GO also actively promoted osteogenesis. The GO-modified porous composite scaffolds synthesized by Şelaru et al increased the expression of RUNX2 and OPN, promoted the deposition of calcifications, and enhanced osteogenic differentiation in vitro, which was consistent with the experimental results of this study. GO-modified concentric microgrooves enhanced the expression of osteogenic differentiation-related genes and proteins, probably because cell pseudopodia extended along the microgrooves, facilitating the preferential alignment of cells along the groove direction, resulting in increased cell–cell interactions .…”

Thermal Behavior of Graphene Oxide Deposited on 3D-Printed Polylactic Acid for Photothermal Therapy: An Experimental–Numerical Analysis

“…Moreover, in comparison to other carbon-based PTCAs, GO offers better water dispersibility than both graphene and reduced graphene oxide (rGO), resulting in low cytotoxicity [ 4 ]. In relation to this latter quality, recent publications [ 1 , 19 , 20 ] have proven GO biocompatibility at certain concentrations.…”

Graphene Oxide/RhPTH(1-34)/Polylactide Composite Nanofibrous Scaffold for Bone Tissue Engineering

“…Many studies have proved that a low concentration of GO can promote cell adhesion and proliferation [ 15 , 17 ] and support the growth and osteogenic differentiation of stem cells in the field of bone tissue engineering [ 20 ]. GO has also had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region [ 21 ]. A total of 0.5 wt.% GO was incorporated into fish gelatin/chitosan/genipin scaffolds, which enhanced the expression of runx2 and opn during osteogenic differentiation [ 21 ].…”

“…GO has also had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region [ 21 ]. A total of 0.5 wt.% GO was incorporated into fish gelatin/chitosan/genipin scaffolds, which enhanced the expression of runx2 and opn during osteogenic differentiation [ 21 ]. Therefore, a good way to improve the mechanical and osteogenic properties of polymers is the incorporation of GO as a nanofiller.…”