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.

Keywords: biomineralization; biopolymer blends; ectopic bone formation; ex vivo analysis; graphene oxide; osteoinduction.

Figure 1

(a) Plotting of the compression modulus of hydrated materials, before implantation; (b) histogram depiction of the wall thickness size domain calculated in CTAn (Bruker); (c) color-highlighted 3D renderings of (*) GCs, (**) GCsGp and (***) GCsGp/GO 0.5% scaffold captured in CTVox.

Figure 2

In vitro osteogenic profile analyses of runx2 (a) and opn (b) gene expression in differentiated 3T3-E1 cells in contact with GCsGp/GO materials with statistical significance ^### p < 0.001; ** p < 0.01; ^#,* p < 0.05; (c) immunohistochemical runx2 and opn expression in differentiated 3T3-E1 cells in contact with GCsGp/GO materials.

Figure 3

Qualitative evaluation of cellular distribution and morphology in GCsGp/GO scaffolds during 7 (A1–A3) and 28 (B1–B3) days of osteogenic differentiation using SEM while the A1’–A3’ and B1’–B3’ subsets depict corresponding close-ups of the areas marked in red squares above; qualitative evaluation of in vitro calcium accumulation in bECM using ARS histological staining at after 7 (Ai–Aiii) and 28 (Bi–Biii) days.

Figure 4

(a) Seric ALP activity 28 days post-implantation of GCs, GCsGp and GCsGp/GO 0.5% wt.% Scaffolds to mice; in vivo osteogenic profile analyses (b) mRNA expression of opn and runx2 four weeks post-implantation (statistical significance ^#,* p < 0.05); (c) confocal microscopy protein expression of opn (red) and runx2 (green) and cell nuclei stained in blue four weeks post-implantation.

Figure 5

Histological analysis of the ectopic bone occurrence in GCs, GCsGp and GCsGp/GO 0.5% wt.% Scaffolds 28 days post-implantation. (a) Representative H&E, Gömöri trichrome and ARS stainings. Scale Bar 20 µm; (b) The analysis of the area of collagen domains according to Gömöri staining indicated that significantly more collagen was secreted within GCsGp/GO 0.5% wt.% Group as opposed to GCs group (* p < 0.001); (c). ARS staining indicates that significantly more calcium mineral deposits are present in the GCsGp/GO 0.5% wt.% group than GCs group (* p < 0.001).

Figure 6

SEM micrographs of GCs, GCsGp and GCsGp/GO 0.5% wt.% scaffolds 28 days post-implantation.

Figure 7

Colorized µCT images of (a) GCs, (b) GCsGp and (c) GCsGp/GO 0.5% wt.% scaffolds explanted 28 days; (*) marks indicate captures whereby the bi-phasic nature of the samples was separately highlighted and (**) marks indicate sectional views of the central morphology of the samples. (d) Charted data correlating mechanical properties and mineral formation based on the constitutional nature of the composites.

Figure 8

(a) FTIR spectra of GCs, GCsGp and GCsGp/GO 0.5% wt.% scaffolds explanted after 28 days; (b) close-up on the 400-600 cm⁻¹ fingerprint domain specific to natural phosphates.

Figure 9

XRD spectra of GCs, GCsGp and GCsGp/GO 0.5% wt.% (a) before implantation and (b) after explanation; (c) crystallinity index of the three scaffold compositions.

Figure 10

Experimental design. (a) Preparation of subcutaneous pocket in the dorsum of mice; (b,c) ectopic subcutaneous implantation of the scaffold; (d) closure of the overlaying skin; (e) scaffolds before implantation; (f) GCsGp/GO 0.5% wt.% scaffold 28 days after subcutaneously implantation to mice.