Introduction: Recently, graphene (G) has shown its potential for applications in tissue engineering[1]-[3]. While most of the studies were conducted on two dimensional membranes of G or graphene oxide (GO), fewer investigations were made on three dimensional G or GO structures which could be readily applied as tissue regeneration scaffolds. The promise of such 3D graphene structures was proved in a recent study[4], where 3D graphene foams prepared by chemical vapor deposition (CVD) promoted osteogenic differentiation. However, the cost of CVD may limit the use of such method. In our study, we prepare a GO/hydroxyapatite (HA) composite homogeneous hydrogel via a simple and low cost hydrothermal treatment of colloidal HA and GO.
Materials and Methods: We start from commercial GO aqueous suspensions (lateral dimensions 0.5–5 µm) and citrate-stabilized HA hydrocolloids, which we prepare according to[5]. GO and HA are mixed to generate homogenous suspensions with a final GO concentration of 4 mg mL−1 and variable mass ratios of HA to GO. The samples are named based on this ratio: for example, G/HA-40 is a gel prepared with an HA/GO feed ratio of 40/60 (wt/wt). The control sample rGO is a reduced GO without HA added.
Results and Discussion: During the hydrothermal treatment, GO is partially deoxygenated and reduced to graphene (rGO), and encapsulates HA nanoparticles inside the hydrogel[6],[7]. SEM image in Figure 1 shows a homogenous distribution of HA on hydrogels with as much as 80% HA. The resulting hydrogel is highly conductive, mechanically robust and can be used as cell culture scaffold for MSCs. Cell culture results in Figure 2 a-c on mouse multipotent MSCs show high cell viability on both rGO and G/HA hydrogels. The cell densities on the two are similar and roughly correspond to 1/2 of that of the polystyrene control (Figure 2d), higher than what observed on CVD graphene foam (4). A closer SEM examination (Figure 2 e and f) shows that cells are well spread and firmly attach to the both rGO and G/HA hydrogels. Cells on g/HA show filamentous extensions which directly interact with the material.
Conclusion: We have successfully developed a strategy to prepare highly homogenous G/HA composite hydrogel materials based on colloidal chemistry and hydrothermal treatment of GO. The strong conductive G/HA hydrogel shows excellent biocompatibility and improved cell affinity compared to previous studies, demonstrating its potential for applications in tissue regeneration. We are currently investigating the osteogenic differentiation of MSCs cultured on these nanocomposites.
Figure 1. SEM images of rGO and G/HA composites at different scales. Uniform distribution of HA NPs is observed in the porous composites
Figure 2. Mouse multipotent mesenchymal stromal cell (MSC) viability and morphology on tissue culture polystyrene (TCP), rGO and G/HA-40 following 2 days of culture. a–c, Live/dead stain shows that the majority of the attached cells are viable (green), and only few cells have died (red). Insets show a higher magnification of live/dead cells. d, The percentage of live cells is as high as ~93% without statistical differences across the three materials (P>0.05), as measured by averaging at least 8 images at 50× magnification from at least 3 specimens. The live cell densities on rGO and G/HA-40 are statistically the same (P>0.05), but significantly lower than on TCP (*, P<0.05). e,f, Cells are well spread on rGO, but more elongated on G/HA-40, with filamentous extensions (* labels) directly interacting with the material.
Canada Research Chair foundation; Natural Sciences and Engineering Research Council (NSERC) of Canada; the Fonds de recherche du Québec Nature et Technologie (FQRNT); Center for Self-Assembled Chemical Structures; McGill Engineering Doctoral Award; Sichuan University Scholarship Fund for; Prof. Thomas Skopek; Dr Mohamed Nur Abdallah; Dr Younan Liu; Prof. Faleh Tamimi Marino; Prof. Bernard Drouin
References:
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